Oligonucleotide encoded chemical libraries, related systems, devices, and methods for detecting, analyzing, quantifying, and testing biologics/genetics

ABSTRACT

This application provides a bead with a covalently attached chemical compound and a covalently attached DNA barcode and methods for using such beads. The bead has many substantially identical copies of the chemical compound and many substantially identical copies of the DNA barcode. The compound consists of one or more chemical monomers, where the DNA barcode takes the form of barcode modules, where each module corresponds to and allows identification of a corresponding chemical monomer. The nucleic acid barcode can have a concatenated structure or an orthogonal structure. Provided are a method for sequencing the bead-bound nucleic acid barcode, for cleaving the compound from the bead, and for assessing biological activity of the released compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application under 35 U.S.C 371of International Application No. PCT/US2021/015550, filed on Jan. 28,2021, which is a continuation-in-part of, claims the benefit of, andclaims priority to co-pending U.S. patent application Ser. No.16/774,871 filed Jan. 28, 2020, the content of which is incorporatedherein by reference in its entirety.

The International Application No. PCT/US2021/015550 is also acontinuation-in-part of, claims the benefit of, and claims priority toco-pending U.S. patent application Ser. No. 16/870,809, filed May 8,2020, the content of which is incorporated herein by reference in itsentirety.

FIELD OF THE DISCLOSURE

The disclosure relates to high-throughput screening using a library ofcompounds, where the compounds are bound to beads, or contained withinbeads, each bead containing multiple copies of one kind of compound,where further, the bead also contains DNA tags that encode the identityor synthetic history of the compound that is contained in or on thebead. The disclosure also relates to high-throughput assays performed inpicowells, where the picowells contain compound-laden beads and assaymaterials. The disclosure further relates to releasing the bead-boundcompounds and screening them for biological activity. Broadly, thedisclosure contemplates assays where beads are used as delivery-vehiclesfor compounds, and methods for creating such compound-laden beads.

The disclosure relates bead-bound compounds, where each compound is madeof one or more monomers belonging to a chemical library. The disclosurealso relates to bead-bound DNA barcodes, that is, to nucleic acids wherethe sequence of each nucleic acid is a code (not related to the geneticcode) refers to one particular chemical library monomer. The disclosurefurther relates to releasing the bead-bound compounds and then screeningthe released compounds for biological activity.

The disclosure also pertains generally to methods for perturbing a cell,or a few cells, with a dose-controlled compound, and analyzing thechange in the state of the cell by RNA and/or protein analysis. Themethods disclosed herein could be applied at the single-cell level, orto a plurality of cells, for the purpose of high throughput screening,target discovery, or diagnostics, and other similar applications.

Further, the present disclosure relates to a computer-implementedmethod, computer-implemented system and computer readable medium, eachfor perturbing a cell and capturing a response of the cell to theperturbation.

BACKGROUND OF THE DISCLOSURE

Combinatorial chemistry, for example, involving split-and-poolchemistry, can be used for synthesizing large amounts of compounds.Compounds made in this way find use in the field of medicinal chemistry,where the compounds can be screened for various biochemical activities.These activities include binding to one or more proteins, where theproteins are known at the time the screening test is performed.Alternatively, the proteins that are bound by a compound being testedare identified only after a binding event is detected. Compounds canalso be screened for their activity of inhibiting or activating a knownprotein (this is not merely screening for a “binding” activity).Alternatively, compounds can be screened for their activity ofinhibiting or activating a cellular function, and where the moleculartargets are not known to the researcher at the time of screening.

The screening of compounds, such as compounds belonging to a hugelibrary of chemicals made by split-and-pool methods, can be facilitatedby conducting screening with an array of many thousands of microwells,nanowells, or picowells. Moreover, screening can be facilitated byproviding a different compound to each picowell by way of a bead, andwhere each bead contains hundreds of copies of the same compound, andwhere the same bead also contains hundreds of copies of a “DNA barcode”that can be used to identify the compound that is attached to the samebead. Moreover, screening of compounds is further facilitated by usingcleavable linkers, where the cleavable linker permits controlled releaseof the compound from the bead, and where the released compound is thenused for biochemical assays or cell-based assays in the same picowell.

Assaying compounds in very small, confined volumes, such as droplets,picowells or microfluidic environments is broadly beneficial, forinstance, due to the low volumes of assay reagents needed, and thereforeneed not be limited to combinatorially generated compounds. Any methodthat can load compounds onto beads, and also allows the compounds to beeluted off the beads at a later time, may be used for deliveringbead-bound compounds to assays in small, confined volumes. The additionof nucleic acid barcodes to the beads allows the identity of thecompound present within the beads to be carried along to the assayvolume. In his manner, very high throughput assays may be performedwithout needing robotics or spatial indexing of compounds withinmicrotiter plates. Millions to billions of compounds may be held withinone small vial, the identity of the compounds tagged on the same bead(with DNA) that contains each individual compound.

A common method for drug discovery involves picking a target of interestand monitoring the interaction of the target protein or enzyme with alarge library of chemical compounds. In many cases, a large number ofinitial hits are found toxic to the body or cross reactive with otherproteins in the body, rendering the target-based selection aninefficient method for drug screening. The need for a pre-selectedtarget is also an inherent limitation, since it requires the biologicalunderpinning of disease to be well-known and understood. Screeningcompounds against an entire organism is a difficult, expensive, and verylow-throughput task.

Conventional phenotypic screening on cells has involved creating modelsof diseased-state cells, contacting the cells with various druglibraries, and monitoring if the disease phenotype is corrected by ameasurable assay. Such screening methods are called phenotypicscreening, as the underlying biological mechanism is not necessarilyunderstood at the beginning, but a measurable, phenotypic change that isindicative of a curative response is considered the relevant metric. Avast number of cell lines and disease models reflecting various baselineand diseased cell states are available today. Also available are largernumbers of compound libraries and biological drugs candidates. Theobvious screening campaign combining different cell models withdifferent drug candidates to look for phenotypic responses is fraughtwith technical limitations as assays are limited to microtiter plateformats and imaging modalities, both of which are severely limited inthroughput.

One method to overcome throughput limitations is to adopthigh-throughput single-cell screening approaches to drug discovery (see,e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016). In theseapproaches, single cells are separated and isolated into compartmentswhere individual assays can be performed on each of the cells. Genomicanalysis via mRNA sequencing of the single cells, e.g., using dropletencapsulation, is a popular method that reveals intricate details thatare hidden in ensemble measurements (see, e.g., Macosko et al., Cell161:1202-1214, 2015 and Ziegenhain et al., Mol Cell 65:631-643, 2017,the disclosures of which are incorporated herein by reference in theirentireties). Present state of the art single-cell analysis platformshave enabled quantitation of mRNA transcripts with single-cellresolution to characterize and fingerprint cells based on theirtranscriptional state. This approach allows for comparison betweentissue samples, extracted from a subject or prepared in an experiment,and examining single-cell transcription, and therefore, proteinexpression states. The measurements of single-cell mRNA by transcriptomesequencing and profiling are important approaches to investigatemolecular mechanisms of not only genealogic phenotypes of cells duringdisease progression, but also drug efficacy, resistances, and discoveryof therapeutic targets (see, e.g., Chu et al., Cell Biol and Toxicol33:83-97, 2017, Wang, Cell Biol Toxicol 32:359-361, 2016, and Wang etal., Cell Biol Toxicol 33:423-427, 2017). The application of single-cellRNA sequencing has been used to define intercellular heterogeneity,evidenced by transcriptomic cell-to-cell variation, which is extremelyrelevant to drug efficacy and specificity, transcriptionalstochasticity, transcriptome plasticity, and genome evolution.Encapsulation in picowells has also been demonstrated (see, e.g.,Gierahn et al., Nat Methods 14:395-398, 2017). Single cell proteinmeasurements are also possible using similar isolation methods (Butniket al., BioRxiv, January 2017, Su et al., Proteomics 17:3-4, 2017).

Despite the rapid rise in high-throughput single-cell RNA-sequencing(RNA-seq) methods, including commercialized versions of automatedplatforms such as the Fluidigm C1, 10× Genomics or 1CellBiO systems, theapplication of single-cell RNA profiling for target agnostichigh-throughput drug screening and target discovery is constrained bythe lack of methods that can efficiently partition different drugs todifferent cells. While incubating cells or tissues under differentperturbations within well plates, followed by single-cell analysis andcomparisons between transcript profiles can be done, the number of drugsthat can be examined is limited by the plate capacity. Further, the needto prepare barcoded mRNA from each sample in isolation and then performcomprehensive RNA profiles for every sample, creates a major bottleneck,as well.

One of the ongoing problems with large scale assays as described hereinis the need in many cases to have a single bead in a single well. Whilethis is typically not a problem when employing a 96 well plate, itbecomes a significant problem when employing an assay device with over100,000 wells. For example, while Vann, et al., U.S. Patent ApplicationPublication No. 2003/0021734, (incorporated herein by reference in itsentirety), discloses a robotic bead dispensing system that is designedto provide for a single bead in a single well, this reference, in fact,states that more than a single bead can be put into a well and requiresvisual confirmation that the wells contain only a single bead (seeparagraph [0131] of Vann et al.).

Herein are described improvements in systems, devices and methods forperturbing a cell and capturing a response of the cell to theperturbation that provide for bead dispensing devices that deposit asingle bead into a single well when such is a required component of alarge scale assay.

SUMMARY OF THE DISCLOSURE

Briefly stated, the present disclosure provides a system for screeningchemical compounds, comprising: (a) A picowell array plate comprising aplurality of picowells, wherein each picowell has a top aperture thatdefines an opening at the top of the picowell, a bottom that is definedby a floor, wherein the top aperture is separated from the floor, andwherein a wall resides in between the top aperture and the floor; (b) Abead disposed in a picowell, wherein the bead comprises a plurality ofsubstantially identical bead-bound DNA barcodes, and a plurality ofsubstantially identical bead-bound compounds, (c) Wherein the beadcomprises a bead-bound DNA barcode that takes the form of either aconcatenated DNA barcode or an orthogonal DNA barcode, and wherein ifthe DNA barcode takes the form of a concatenated DNA barcode theconcatenated DNA barcode is made by a method that: (i) Uses clickchemistry, or (ii) Uses a repeating cycle of steps, wherein therepeating cycle of steps comprises using a splint oligonucleotide(splint oligo) that is capable of hybridizing to a partially madebead-bound DNA barcode, and wherein the hybridizing is mediated by anannealing site on the splint oligo and a corresponding, complementaryannealing site in the partially made bead-bound DNA barcode, wherein theannealed splint oligo is used as a template for extending the partiallymade DNA barcode using DNA polymerase, and wherein the splint oligocontains bases that are complementary to a DNA barcode module that is tobe polymerized to the partially made DNA barcode, (d) Wherein each oneof the plurality of substantially identical bead-bound compoundscomprises one or more chemical library monomers, and wherein eachbead-bound DNA barcode module identifies a corresponding chemicallibrary monomer, wherein the term “compound” is used to refer to acompleted product that comprises one or more chemical library members,and wherein the completed DNA barcode identifies the compound.

The floor of a microwell, nanowell, or picowell, need not be flat. Thefloor may be curved as in the manner of the bottom of a glass test tubeor metal centrifuge tube. Also, the floor may be conical-shaped, as inconical centrifuge tubes. The floor may be flat but with notches, forexample, notches that facilitate motion of an assay solution or cellculture solution in the vicinity of the bottom of any bead that issitting in the picowell. In flat-floor embodiments, the present systemand methods can require a flat floor.

The concatenated DNA barcode can be made entirely by methods of organicchemistry, for example, by click chemistry. Also, the orthogonal DNAbarcode can be made entirely by methods of organic chemistry, forexample, comprising click chemistry.

What is also provided is the above system, further comprising aplurality of caps, each cap capable of fitting into the opening of adifferent picowell, and each cap capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each capable ofminimizing or preventing leakage of fluid that is inside of thepicowell.

Moreover, what is embraced is the above system, wherein the concatenatedDNA barcode is made by a method that uses: (i) Both click chemistry andthe repeating cycle of steps that uses the splint oligo; (ii) Both clickchemistry and chemical methods that are not click chemistry methods;(iii) Only click chemistry; or (iv) Only the repeating cycle of stepsthat uses the splint oligo. For this particular embodiment the“concatenated DNA barcode” in question does not include any chemicalcoupler that is used to couple a nucleic acid directly to the bead.

In a spherical cap embodiment, what is provided is the above system,further comprising a plurality of spherical caps, wherein each cap iscapable of fitting into the aperture of a picowell wherein the apertureis circular, and each cap is capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each cap iscapable of minimizing or preventing leakage of fluid that is inside ofthe picowell.

In a response element embodiment, what is provided is the above system,wherein the at least one bead disposed in the at least one picowellcomprises at least one response capture element that is coupled to saidat least one bead. Also, what is contemplated is the above system,wherein the at least one bead disposed in at least one picowellcomprises at least one response capture element that is coupled to saidat least one bead, wherein the at least one response capture elementcomprises: (a) Poly(dT) or (b) An exon-targeting RNA probe.

Also contemplated is the above system, wherein the DNA barcode is eithera concatenated DNA barcode or an orthogonal DNA barcode, and wherein theDNA barcode comprises one or more DNA barcode modules, wherein each ofthe one or more DNA barcode modules encodes information that identifiesa chemical library monomer, and wherein the concatenated DNA barcode orthe orthogonal DNA barcode further includes one or both of: (a) One ormore functional nucleic acids; and (b) One or more nucleic acids thatencode information of a type other than the identity of a chemicallibrary monomer.

The following discloses “consists of only” embodiments and “comprises”embodiments, as it applies to the number of bead-bound DNA barcodemodules that make up a DNA barcode. What is provided is embodimentswhere the DNA barcode consists of only one DNA barcode module, or onlytwo DNA barcode modules, or contains only three DNA barcode modules, oronly four DNA barcode modules, and so on, or where the DNA barcodecomprises at least one DNA barcode module, or comprises at least two DNAbarcode modules, or comprises at least three DNA barcode modules, orcomprises at least four DNA barcode modules, and so on,

What is also embraced, is a system wherein the bead-bound concatenatedDNA barcode comprises: (i) a 1^(st) DNA barcode module; or (i) a 1^(st)DNA barcode module, a 1^(st) annealing site, and a 2^(nd) DNA barcodemodule; or (ii) a 1^(st) DNA barcode module, a 1^(st) annealing site, a2^(nd) DNA barcode module, a 2^(nd) annealing site, and a 3^(rd) DNAbarcode module; or (iii) a 1^(st) DNA barcode module, a 1^(st) annealingsite, a 2^(nd) DNA barcode module, a 2^(nd) annealing site, a 3^(rd) DNAbarcode module, a 3^(rd) annealing site, and a 4^(th) DNA barcodemodule; or (iv) a 1^(st) DNA barcode module, a 1^(st) annealing site, a2^(nd) DNA barcode module, a 2^(nd) annealing site, a 3^(rd) DNA barcodemodule, a 3^(rd) annealing site, a 4^(th) DNA barcode module, a 4^(th)annealing site, and a 5^(th) DNA barcode module; or (v) a 1^(st) DNAbarcode module, a 1^(st) annealing site, a 2^(nd) DNA barcode module, a2^(nd) annealing site, a 3^(rd) DNA barcode module, a 3^(rd) annealingsite, a 4^(th) DNA barcode module, a 4^(th) annealing site, a 5^(th) DNAbarcode module, a 5^(th) annealing site, and a 6^(th) DNA barcodemodule.

Moreover, what is contemplated is the above system, further comprising aprimer binding site capable of binding a DNA sequencing primer, whereinsaid primer binding site is capable of directing sequencing of one ormore of the 1^(st) DNA barcode module, the 2^(nd) DNA barcode module,the 3^(rd) DNA barcode module, the 4^(th) DNA barcode module, the 5^(th)DNA barcode module, or the 6^(th) DNA barcode module, and wherein theprimer binding site is situated 3-prime to the 1^(st) DNA barcodemodule, 3-prime to the 2^(nd) DNA barcode module, 3-prime to the 3^(rd)DNA barcode module, 3-prime to the 4^(th) DNA barcode module, 3-prime tothe 5^(th) DNA barcode module, or 3-prime to the 6^(th) DNA barcodemodule, or wherein the primer binding site is situated in between the1^(st) and 2^(nd) DNA barcode modules, or is situated in between the2^(nd) and 3^(rd) DNA barcode modules, or is situated in between the3^(rd) and 4^(th) DNA barcode modules, or is situated between the 4^(th)and 5^(th) DNA barcode modules, or is situated between the 5^(th) and6^(th) DNA barcode modules.

Additionally, what is provided is the above system, wherein the primerbinding site is situated in between the 1^(st) and 2^(nd) DNA barcodemodules, or is situated in between the 2^(nd) and 3^(rd) DNA barcodemodules, or is situated in between the 3^(rd) and 4^(th) DNA barcodemodules, or is situated between the 4^(th) and 5^(th) DNA barcodemodules, or is situated between the 5^(th) and 6^(th) DNA barcodemodules. In embodiments relating to the position of a primer bindingsite, relative to upstream DNA barcode modules and relative todownstream DNA barcode modules, what is provided is the above system,wherein a primer binding site is situated in between each and every pairof successive DNA barcode modules.

Furthermore, what is provided is the above system, wherein the beadcomprises a DNA barcode that is an orthogonal DNA barcode, wherein thebead comprises an external surface, and wherein the orthogonal DNAbarcode comprises: (a) A first nucleic acid that comprises a first DNAbarcode module and an annealing site for a sequencing primer, whereinthe first nucleic acid is coupled to the bead at a first position, (b) Asecond nucleic acid that comprises a second DNA barcode module and anannealing site for a sequencing primer, wherein the second nucleic acidis coupled to the bead at a second position, and (c) A third nucleicacid that comprises a third DNA barcode module and an annealing site fora sequencing primer, wherein the second nucleic acid is coupled to thebead at a third position, and wherein the first, second, and thirdposition on the bead are each located at different location on thebead's external surface.

In encoding embodiments, what is provided is the above system, whereinthe DNA barcode comprises one or more nucleic acids that do not identifyany chemical library monomer but that instead identify: (a) The class ofchemical compounds that is cleavably attached to the bead; (b) The stepnumber in a multi-step pathway of organic synthesis; (c) The date thatthe bead-bound compound was synthesized; (d) The disease that thebead-bound compound is intended to treat; (e) The cellular event thatthe bead-bound compound is intended to stimulate or inhibit; or (f) Thereaction conditions that were used to couple a given chemical librarymonomer to the bead.

In linker embodiments, what is provided is the above system, whereineach of the plurality of substantially identical bead-bound compounds iscoupled to the bead by way of a cleavable linker. Also provided is theabove system, wherein each of the plurality of substantially identicalbead-bound compounds is coupled to the bead by way of a light-cleavablelinker. Also provided is the above system, wherein each of the pluralityof substantially identical bead-bound compounds is coupled to the beadby way of a non-cleavable linker.

In TentaGel® embodiments, what is provided is the above system, whereinthe at least one bead comprises grafted copolymers consisting of a lowcrosslinked polystyrene matrix on which polyethylene glycol (PEG) isgrafted.

In release-monitor embodiments, the present disclosure provides theabove system, wherein at least one picowell contains a release-monitorbead, and does not contain any other type of bead,

wherein the release-monitor bead comprises a bead-bound quencher and abead-bound fluorophore, wherein the bead-bound quencher is quenchinglypositioned in the immediate vicinity of the bead-bound fluorophore andcapable of quenching at least 50% (or at least 60%, or at least 70%, orat least 80%, or at least 90%, or at least 95%, or at least 99%, or atleast 99.5%, or at least 99.9%) of the fluorescence of the bead-boundfluorophore, and wherein the bead-bound fluorophore is bound by way of afirst light-cleavable linker, wherein the picowell containing therelease-monitor bead is a first picowell, wherein the first picowellcontains a first solution, wherein exposing the first picowell tocleaving conditions is capable of severing the light-cleavable linkerand releasing the fluorophore into the first solution of the firstpicowell, wherein the exposing results in the fluorophore diffusingthroughout the first solution in the first picowell, and wherein afluorescent signal acquired by shining light on the first picowell thatcontains the first solution comprising diffused fluorophore allows theuser to use the fluorescent signal to calculate the percent release ofthe bead-bound fluorophore from the release-monitor bead resulting in avalue for the calculated percent release, and wherein a second picowellcontains a bead-bound compound coupled with the same type oflight-cleavable linker as the first light-cleavable linker, and whereinthe second picowell contains a second solution,

and wherein the value for the calculated percent release from therelease-monitor bead in the first picowell allows calculation of theconcentration of the released compound in the second solution of thesecond picowell.

In embodiments relating to identity of all of the compounds bound to agiven bead, or relating to identity of all of the DNA barcodes bound toa given bead, what is provided is the above system, wherein the at leastone bead comprises a plurality of substantially identical bead-bound DNAbarcodes, wherein the plurality is between 10 million to 100 millioncopies of the substantially identical bead-bound DNA barcodes. Alsoprovided is the above system, wherein the at least one bead comprises aplurality of substantially identical bead-bound compounds, where whereinthe plurality is between 10 million to 100 million copies of thesubstantially identical bead-bound compounds.

In embodiments relating to cells (e.g., mammalian cells, cancer cells,bacterial cells), what is provided is the above system, wherein at leastone picowell comprises at least one cell, wherein the plurality ofsubstantially identical bead-bound compounds are bound to the at leastone bead by way of a cleavable linker, and wherein cleaving thecleavable linker releases the bead-bound compound from the bead toproduce a released compound, and wherein the released compound iscapable of contacting the at least one cell. In other cell embodiments,what is provided is the above system, wherein at least one picowellcomprises at least one cell, wherein the plurality of substantiallyidentical bead-bound compounds are bound to the at least one bead by wayof a cleavable linker, and wherein cleaving the cleavable linkerreleases the bead-bound compound from the bead to produce a releasedcompound, and wherein the released compound is capable of contacting theat least one cell, and wherein the at least one cell is: (i) a mammaliancell that is not a cancer cell, (ii) a mammalian cancer cell, (iii) adead mammalian cell, (iv) an apoptotic mammalian cell, (v) a necroticmammalian cell, (vi) a bacterial cell, (vii) a plasmodium cell, (vii) acell that is metabolically active but has a cross-linked genome and isunable to undergo cell division, or (ix) a mammalian cell that isinfected with a virus.

In device embodiments, what is provided is the above system, whereineach picowell has a top aperture that defines an opening at the top ofthe picowell, a bottom that is defined by a floor, wherein the topaperture is separated from the floor, and wherein a wall resides inbetween the top aperture and the floor, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a truncated cone, and wherein the aperture has a first diameter, thefloor has a second diameter, and wherein the first diameter is greaterthan the second diameter.

In other device-related embodiments, what is provided is the abovesystem, wherein each picowell has a top aperture that defines an openingat the top of the picowell, a bottom that is defined by a floor, whereinthe top aperture is separated from the floor, and wherein a wall residesin between the top aperture and the floor, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a truncated cone, and wherein the aperture has a first diameter, thefloor has a second diameter, and wherein the first diameter is greaterthan the second diameter, further comprising a cap that snuggly fitsinto the aperture, wherein the aperture is comprised by a polymer havinga greater durometer (harder) and wherein the cap is made of a polymerhaving a lesser durometer (softer), and wherein the relative durometersof the cap and aperture allow the cap to be reversibly and snuggly fitinto the aperture, and wherein the cap is: (i) a cap intended only toplug the picowell and prevent leakage, (ii) a cap that is a passive capand that is capable of absorbing metabolites that are released by acell, in the situation where a cell in a cell medium is cultured in thepicowell, (iii) a cap that is an active cap, and that takes the form ofa bead that comprises a plurality of essentially identical compounds,and wherein each of the plurality of essentially identical compounds iscoupled to the bead with a cleavable linker; (iv) a cap that is anactive cap, and that takes the form of a bead that comprises a pluralityof identical reagents, and wherein each of the plurality of essentiallyidentical reagents is coupled to the bead with a cleavable linker. Alsoprovided is the above system, wherein the cap is spherical, or whereinthe cap is non-spherical.

In mat embodiments, the above system comprises a picowell array platecomprising an upper generally planer surface, a plurality of picowells,wherein each picowell has a top aperture that defines an opening at thetop of the picowell, a bottom that is defined by a floor, wherein thetop aperture is separated by a wall from the floor, and wherein the wallresides in between the top aperture and the floor, and optionally, abead disposed in at least one of said plurality of picowells, whereinthe bead comprises a plurality of substantially identical bead-bound DNAbarcodes, and a plurality of substantially identical bead-boundcompounds, wherein the picowell array plate further comprises a mat thatis capable of securely covering the opening at the top of at least oneor all of the plurality of picowells, or that is actually securelycovering the opening at the top of at least one or all of the pluralityof picowells, wherein the securely covering is reversible, wherein themat optionally comprises one or all of: (a) An absorbent surface that,when positioned in contact with the upper generally planer surface ofthe picowell array plate, is capable of absorbing any metabolites,biochemicals, or proteins that may be comprised by one or more of theplurality of picowells, (b) An adhesive surface that is capable ofmaintaining reversible adhesion to the top generally planer surface ofthe picowell array plate.

In biochemical assay embodiments, what is embraced is the above system,that includes at least one picowell, wherein the at least one picowellcomprises a bead that comprises a plurality of substantially identicalcompounds and a plurality of substantially identical barcodes, whereinthe at least one picowell comprises an assay medium that includescereblon E3 ubiquitin ligase, a substrate of cereblon E3 ubiquitinligase such as Ikaros or Aiolos, and wherein the system is capable ofscreening for compounds that activate cereblon's E3 ubiquitin ligaseactivity, and are thereby capable of reducing intracellularconcentrations of Ikaros or Aiolos.

In another biochemical assay embodiment, what is contemplated is theabove system, that includes at least one picowell, wherein the at leastone picowell comprises a bead that comprises a plurality ofsubstantially identical compounds and a plurality of substantiallyidentical barcodes, wherein the at least one picowell comprises an assaymedium that includes MDM2 E3 ubiquitin ligase, a substrate of MDM2 E3ubiquitin ligase such as p53, and wherein the system is capable ofscreening for compounds that activate MDM2's E3 ubiquitin ligaseactivity, and thereby capable of increasing the intracellularconcentrations of p53.

In more barcoding embodiments, what is provided is the above system,wherein the DNA barcode comprises one or more nucleic acids that do notencode any chemical monomer but that instead identify one or more of:(a) The class of chemical compounds that is cleavably attached to thebead; (b) The step in a multi-step pathway of organic synthesis, whereina bead-bound nucleic acid corresponds to a given chemical monomer thatis used to make a bead-bound compound, and wherein the bead-boundnucleic acid that corresponds to a given chemical monomer identifiesthat chemical monomer; (c) The date that the bead-bound compound wassynthesized; (d) The disease that the bead-bound compound is intended totreat; (e) The cellular event that the bead-bound compound is intendedto stimulate or inhibit.

In embodiments that lack any headpiece, what is provided is the abovesystem, wherein the at least one bead comprises a plurality ofsubstantially identical bead-bound compounds and also comprises aplurality of substantially identical bead-bound DNA barcodes, andwherein there does not exist any headpiece that links any of thebead-bound compounds to any of the bead-bound DNA barcodes.

Moreover, what is contemplated is the above system, wherein at least70%, at least 80%, at least 90%, at least 95%, or at least 98% of thesubstantially identical bead-bound DNA barcodes have an identicalstructure. Additionally, what is contemplated is the above system,wherein at least 70%, at least 80%, at least 90%, at least 95%, or atleast 98% of the substantially identical bead-bound compounds have anidentical structure.

Furthermore, what is supplied is the above system, wherein theconcatenated DNA barcode comprises at least one nucleic acid that is aDNA barcode module, or the above system, wherein the concatenated DNAbarcode comprises only one nucleic acid that is a DNA barcode module.

In sequencing primer annealing site embodiments, what is provided is theabove system, wherein the concatenated DNA barcode comprises at leastone nucleic acid that is a DNA barcode module, and at least onefunctional nucleic acid that: (a) Is capable of being used as anannealing site for a sequencing primer, (b) Is capable of forming ahairpin structure, and wherein the hairpin structure comprises asequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure wherein the bend is 5-prime to thesequencing primer and is 3-prime to the annealing site for thesequencing primer, or (c) Is a spacer nucleic acid.

In other sequencing primer embodiments, what is provided is the abovesystem, wherein the orthogonal DNA barcode contains a plurality of DNAbarcode modules, wherein each of the DNA barcode modules is coupled to adifferent site on the bead either directly or via a linker, and whereineach of the plurality of DNA barcode modules contains at least onefunctional nucleic acid that: (a) Is capable of being used as anannealing site for a sequencing primer, (b) Is capable of forming ahairpin structure, and wherein the hairpin structure comprises asequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure wherein the bend is 5-prime to thesequencing primer and is 3-prime to the annealing site for thesequencing primer, or (c) Is a spacer nucleic acid.

In embodiments that recite functional language about splint oligos, whatis provided is a bead comprising a concatenated DNA barcode, wherein theconcatenated DNA barcode comprises: (a) a first DNA barcode module and afirst annealing site for a first splint oligonucleotide (splint oligo),wherein the splint oligo comprises three nucleic acids, wherein thethree nucleic acids are: a nucleic acid that is a hybridizing complementto the first annealing site, a nucleic acid that is a hybridizingcomplement to a 2^(nd) DNA barcode module, and a nucleic acid that is a2^(nd) annealing site, and (b) a second DNA barcode module and a 2ndannealing site for a second splint oligo, wherein the second splintoligo comprises three nucleic acids, wherein the three nucleic acidsare: a nucleic acid that is a hybridizing complement to the 2ndannealing site, a nucleic acid that is a 3rd DNA barcode module, and anucleic acid that is a 3rd annealing site.

In another embodiment that contains functional language relating tosplint oligos, what is provided is the above bead, further comprising: athird DNA barcode module and a 3rd annealing site for a third splintoligo, wherein the third splint oligo comprises three nucleic acids,wherein the three nucleic acids are: a nucleic acid that is ahybridizing complement to the 3rd annealing site, a nucleic acid that isa 4^(th) DNA barcode module, and a nucleic acid that is a 4th annealingsite.

Moreover, in yet another embodiment containing functional languagerelating to splint oligos, what is provided is the above bead, furthercomprising one or more of: (i) a fourth DNA barcode module and a 4thannealing site for a fourth splint oligo, wherein the fourth splintoligo comprises three nucleic acids, wherein the three nucleic acidsare: a nucleic acid that is a hybridizing complement to the 4thannealing site, a nucleic acid that is a 5^(th) DNA barcode module, anda nucleic acid that is a 5th annealing site, (ii) a response captureelement, (iii) a release monitor.

In linker embodiments, what is embraced is the above bead, wherein theconcatenated DNA barcode is coupled to the bead, but is: (i) not coupledto the bead by way of any photocleavable linker, (ii) not coupled to thebead by any enzymatically cleavable linker; or (iii) not coupled to thebead by any kind of cleavable linker.

In an embodiment relating to distinct coupling positions, what isprovided is the above bead, wherein the concatenated DNA barcode iscoupled to a first position on the bead, wherein the bead also comprisesa compound that is coupled to a second position on the bead, and whereinthe first position is not the same as the second position.

In surface embodiments (interior and exterior surfaces), what isprovided is the above bead, wherein the bead comprises an exteriorsurface and an interior surface, wherein the bead comprises at least10,000 substantially identical concatenated DNA barcodes that arecoupled to the bead, and wherein at least 90% of the at least 10,000substantially identical concatenated DNA barcodes are coupled to theexterior surface.

In exclusionary embodiments that can distinguish the present disclosurefrom other embodiments, what is provided is the above bead, that is doesnot comprise any polyacrylamide, and wherein the concatenated DNAbarcode: (i) Does not include any nucleic acid that is a promoter; (ii)Does not include any nucleic acid that is polyA; or (iii) Does notinclude any nucleic acid that is a promoter and does not include anynucleic acid that is polyA.

In release-monitor bead embodiments, the present disclosure supplies arelease-monitor bead that is capable of functioning in an aqueousmedium, wherein the release-monitor bead comprises a bead-bound quencherand a bead-bound fluorophore, wherein the bead-bound quencher isquenchingly positioned in the immediate vicinity of the bead-boundfluorophore and capable of quenching at least 50% of the fluorescence ofthe bead-bound fluorophore, and wherein the bead-bound fluorophore isbound by way of a first light-cleavable linker, wherein the picowellcontaining the release-monitor bead is a first picowell, wherein thefirst picowell contains a first solution, wherein exposing the firstpicowell to cleaving conditions is capable of severing thelight-cleavable linker and releasing the fluorophore into the firstsolution of the first picowell, wherein the exposing results in thefluorophore diffusing throughout the first solution in the firstpicowell, and wherein a fluorescent signal acquired by shining light onthe first picowell that contains the first solution comprising diffusedfluorophore allows the user to use the fluorescent signal to calculatethe percent release of the bead-bound fluorophore from therelease-monitor bead resulting in a value for the calculated percentrelease, and wherein a second picowell contains a bead-bound compoundcoupled with the same type of light-cleavable linker as the firstlight-cleavable linker, and wherein the second picowell contains asecond solution, and wherein the value for the calculated percentrelease from the release-monitor bead in the first picowell allowscalculation of the concentration of the released compound in the secondsolution of the second picowell. In other release-monitor embodiments,what is provided is a release-monitor bead wherein the fluorophore isTAMRA and wherein the quencher is QSY7, and a release-monitor bead thathas the structure shown in FIG. 9 , and a release-monitor bead of thathas the structure shown in FIG. 10 , and a release-monitor bead, whereinthe capable of quenching is at least 90%, at least 98%, at least 99%, orat least 99.9%.

In a methods of manufacture embodiment, what is embraced is a method forsynthesizing a release-monitor bead, wherein the release-monitor beadcomprises a bead, a quencher, a fluorophore, and a photocleavable linkerthat couples the fluorophore to the bead, the method comprising, in thisorder, (i) Providing a resin, (ii) Coupling a lysine linker to theresin, wherein the reagent containing the lysine linker isL-Fmoc-Lys(4-methyltrityl)-OH, (iii) Removing the Fmoc protecting group,(iv) Coupling the quencher using a reagent that isquencher-N-hydroxysuccinimide (quencher-NHS) as the source of quencher,(v) Removing the 4-methyltrityl protecting group using a reagentcomprising trifluoroacetic acid, (vi) Coupling a photocleavable linkerto the epsilon amino group of lysine, wherein the photocleavable linkeris provided by a reagent that is, Fmoc-photocleavable linker-OH, (vii)Coupling the fluorophore. Also provided is the above embodiment, butwithout regard to the ordering of steps. In other methods embodiments,what is provided is the above method wherein the fluorophore is TAMRAand wherein the quencher is QSY7.

In methods relating to the utility of release-monitor bead, what isprovided is a method for controlling the concentration of a compound ina solution that resides in a picowell, wherein the method is applied toa bead-bound compound in a picowell, wherein the picowell contains asolution, and wherein the bead-bound compound is coupled to the bead byway of a cleavable linker, the method comprising: (a) Exposing thebead-bound compound to a condition that effects cleavage of thecleavable linker and releases the bead-bound compound from the bead togenerate a released compound, wherein release is followed by diffusionor dispersion of the released compound in the solution to result in asubstantially uniform concentration of the compound in the solution, (b)Wherein the condition comprises light that is capable of cleaving thecleavable linker, (c) Wherein the condition is adjusted to produce adetermined concentration of the substantially uniform concentration, and(d) Wherein the determined concentration is made with regard to theconcentration of a released fluorophore that is released by from abead-bound release-monitor. Provided also, is the above method, whereinthe condition is adjusted by adjusting one or more of the wavelength ofthe light, the intensity of the light, and by the duration of lightexposure, and the above method, wherein the concentration of a releasedfluorophore that is released from a bead-bound release-monitor isdetermined at the same time as effecting release of the bead-boundcompound from the bead to generate a released compound, and the abovemethod, wherein the concentration of a released fluorophore that isreleased from a bead-bound release-monitor is determined at a timesubstantially before effecting release of the bead-bound compound fromthe bead to generate a released compound.

The term “determined” can mean a concentration that is predetermined anddecided upon as being a desired concentration, prior to exposing thebead to light. Also, the term “determined” can mean a concentration thatis decided upon in “real time,” that is, a concentration that is decidedupon at the same time as the exposing the bead to light.

In cap embodiments, what is embraced is a cap in combination with apicowell plate that comprises a plurality of picowells, wherein the capis capable of use with the picowell plate that comprises a plurality ofpicowells, wherein each of the plurality of picowells is definable by anaperture, a floor, and a wall, wherein the wall is defined by theaperture on top and the floor on the bottom, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a surface of a truncated cone, and wherein the aperture has a firstdiameter, the floor has a second diameter, and wherein the firstdiameter is greater than the second diameter,

wherein the cap is a spherical cap that is capable of snuggly fittinginto the aperture, wherein the aperture is comprised by a polymer havinga greater durometer (harder) and wherein the cap is made of a polymerhaving a lesser durometer (softer), and wherein the relative durometersof the cap and aperture allow the spherical cap to be reversibly andsnuggly fit into the aperture, and wherein the cap is: (i) capable ofplugging the picowell and preventing leakage, (ii) a passive cap andthat is capable of absorbing metabolites that are released by a cell, inthe situation where a cell in a cell medium is cultured in the picowell,(iii) an active cap that takes the form of a bead that comprises aplurality of essentially identical compounds, and wherein each of theplurality of essentially identical compounds is coupled to the bead witha cleavable linker, and wherein cleavage of the cleavable linkerreleases at least some of the plurality of compounds from the bead, (iv)an active cap that takes the form of a bead that comprises a pluralityof identical reagents, and wherein each of the plurality of essentiallyidentical reagents is coupled to the bead with a cleavable linker, andwherein cleavage of the cleavable linker releases at least some of theplurality of reagents from the bead.

In porous cap embodiments, what is provided is a plurality of porouscaps in combination with a picowell plate and a solid polymer coating,wherein each of the plurality of porous caps comprises an upper surfaceand a lower surface, wherein the picowell plate comprises a plurality ofpicowells, wherein at least one porous cap contacts a picowell andreversibly and snuggly fits into the picowell, wherein the picowellplate and each of the upper surfaces of the plurality of porous caps iscovered with a solid polymer coating, wherein the solid polymer coatingcontacts at least some of the upper surface of each cap and isadhesively attached to said at least some of the upper surface, andwherein, (i) Each of the plurality of picowells is capable of holding anaqueous solution, wherein products of a reaction are generated in thesolution, and wherein at least some of the products are absorbed by thelower surface of each of the plurality of porous caps, (ii) Wherein asolution of a polymerizable reagent that capable of polymerization ispoured over the plurality of porous caps in combination with thepicowell plate, and wherein the polymerizable reagent is polymerized toform a substantially planar surface that coats substantially all of thetop surface of the picowell plate, thereby fixing the polymerizedreagent to each of the plurality of porous caps, and (iii) Wherein allof the plurality of porous caps are removable by the act of peeling fromthe plurality of picowells, wherein adhesion is maintained between theplurality of porous caps and the polymerized reagent, resulting in anarray of adhering caps partly with the upper surface of each cap isembedded in the polymerized reagent and the lower surface of each cap isaccessible for analysis of any absorbed reaction product.

This provides a methods of manufacture embodiment, for using splintoligos to guide the enzymatic synthesis of a DNA barcode. What isprovided is a method for making a bead-bound concatenated DNA barcode,wherein the bead-bound concatenated DNA barcode comprises a plurality ofDNA barcode modules, and optionally one or more functional nucleicacids, and optionally one or more identity-encoding nucleic acids thatencode the identity of something other than the identity of a chemicallibrary monomer, the method comprising: (a) The step of providing a beadwith a coupled polynucleotide that comprises a 1^(st) DNA barcode moduleand a 1^(st) annealing site, wherein the 1^(st) annealing site iscapable of hybridizing with a first splint oligonucleotide (splintoligo), the first splint oligo being capable of serving as a templatefor DNA polymerase to catalyze the polymerization to the coupledpolynucleotide, nucleotides that are complementary to those of thehybridized first splint oligo, wherein the polymerized nucleotides thatare complementary to those of the hybridized first splint oligofollowing polymerization comprise a bead-bound 2^(nd) DNA barcode moduleand a 2^(nd) annealing site; (b) The step of providing said bead with acoupled polynucleotide with said first splint oligo, and allowing saidfirst splint oligo to hybridize with said coupled polynucleotide; (c)The step of adding a DNA polymerase and deoxynucleotide triphosphates(dNTPs) and allowing the DNA polymerase to catalyze polymerization ofsaid dNTPs to the coupled polynucleotide, wherein the coupledpolynucleotide has a free 3′-terminus and wherein the polymerization isto the free 3′-terminus, (d) The step of washing away the first splintoligo. Also contemplated is the above method, wherein the first splintoligo comprises a 1^(st) annealing site, a 2^(nd) DNA barcode module,and a 2^(nd) annealing site.

In further methods of manufacture embodiments, what is provided is theabove method, wherein the first splint oligo comprises a 1^(st)annealing site, a 2^(nd) DNA barcode module, a 2^(nd) annealing site,and a nucleic acid encoding a 1^(st) sequencing primer annealing site,wherein the 1^(st) sequencing primer annealing site is capable ofhybridizing to a sequencing primer resulting in a hybridized sequencingprimer, and wherein the hybridized sequencing primer is capable ofdirecting the sequencing of the 2^(nd) DNA barcode module and the 1^(st)DNA barcode module.

Moreover, what is contemplated is the above method, wherein the firstsplint oligo, the DNA polymerase, and the dNTPs are all added at thesame time, or wherein the first splint oligo, the DNA polymerase, andthe dNTPs are each added at separate times.

Regarding interior versus exterior locations on a bead, what is providedis the above method, wherein the bead comprises an exterior location andan interior location, and wherein the bead-bound concatenated DNAbarcode is coupled to the bead at locations that are substantially onthe exterior of the bead and sparingly at interior locations of thebead, and wherein the bead also comprises a plurality of coupledcompounds wherein all of the plurality of coupled compounds havesubstantially an identical structure, when compared to each other, andwherein the bead is comprised substantially of a hydrophobic polymer.

In further methods embodiments, what is provided is the above method,further comprising: (a) The step of providing a bead with a coupledfirst longer polynucleotide that comprises a 1^(st) DNA barcode module,a 1^(st) annealing site, a 2^(nd) DNA barcode, and a 2^(nd) annealingsite, wherein the 2^(nd) annealing site is capable of hybridizing with asecond splint oligo, the second splint oligo being capable of serving asa template for DNA polymerase to catalyze the polymerization to thecoupled first longer polynucleotide, nucleotides that are complementaryto those of the hybridized second splint oligo, wherein the polymerizednucleotides that are complementary to those of the hybridized secondsplint oligo following polymerization comprise a bead-bound 3^(rd) DNAbarcode module and a 3^(rd) annealing site; (b) The step of providingsaid bead with a coupled polynucleotide with said 2^(nd) splint oligo,and allowing said 2^(nd) splint oligo to hybridize with said coupledfirst longer polynucleotide; (c) The step of adding a DNA polymerase anddeoxynucleotide triphosphates (dNTPs) and allowing DNA polymerase tocatalyze polymerization of said dNTPs to the coupled longerpolynucleotide, wherein the coupled longer polynucleotide has a free3′-terminus and wherein the polymerization is to the free 3′-terminus,(d) The step of washing away the second splint oligo.

This relates to the consecutive numbering of the first DNA barcodemodule, the second DNA barcode module, the third DNA barcode module, andso on, for the manufacture of the entire DNA barcode. This also relatesto repeating the cycle of methods steps, over and over and over, in themanufacture of the entire DNA barcode. What is provided is the abovemethod, wherein each of said plurality of DNA barcode modules isidentified or named by a number, the method further comprisingreiterating the recited steps, where for a first reiteration, the nameof the DNA barcode module is increased by adding one number to theexisting name, the name of the annealing site is increased by adding onenumber to the existing name, and the name of the splint oligo isincreased by adding one number to the name of the existing distalterminal DNA barcode module, and the name of the “first longerpolynucleotide” is changed by adding one number to the existing name,wherein the comprising reiterating the recited steps is one reiteration,or two reiterations, or three reiterations, or four reiterations, orfive reiterations, or more than five reiterations, or more than tenreiterations.

Also contemplated is the above method, that comprises a plurality ofsplint oligos, wherein each splint oligo comprises a sequencing primerannealing site, wherein the sequencing primer annealing site is capableof hybridizing to a sequencing primer resulting in a hybridizedsequencing primer, and wherein the hybridized sequencing primer iscapable of directing the sequencing of the at least one bead-bound DNAbarcode module and at least one bead-bound DNA barcode module.

This concerns embodiments relating to splint oligos that guides DNApolymerase to synthesize functional nucleic acids and various types ofinformative nucleic acids. What is provided is the above method, whereinat least one splint oligo comprises a functional nucleic acid, orwherein at least one splint oligo encodes information other thaninformation on a chemical library monomer. What is provided is the abovemethod, further comprising the step of coupling of at least one DNAbarcode module by way of click chemistry, wherein the step does not useany splint oligo.

Briefly stated, the present disclosure provides a system for screeningchemical compounds, comprising: (a) A picowell array plate comprising aplurality of picowells, wherein each picowell has a top aperture thatdefines an opening at the top of the picowell, a bottom that is definedby a floor, wherein the top aperture is separated from the floor, andwherein a wall resides in between the top aperture and the floor; (b) Atleast one bead disposed in at least one picowell, wherein the at leastone bead comprises a plurality of substantially identical bead-bound DNAbarcodes, and a plurality of substantially identical bead-boundcompounds, (c) Wherein the at least one bead comprises a DNA barcodethat takes the form of either a concatenated DNA barcode or anorthogonal DNA barcode, and wherein if the DNA barcode takes the form ofa concatenated DNA barcode the concatenated DNA barcode is made using amethod that: (i) Uses click chemistry, or (ii) Uses a repeating cycle ofsteps, wherein the steps in the repeating cycle comprise using a splintoligo for annealing to a partially made DNA barcode, wherein theannealed splint oligo is used as a template for extending the partiallymade DNA barcode using DNA polymerase, and wherein the splint oligocontains bases that are complementary to a DNA barcode module that is tobe polymerized to the partially made DNA barcode.

In another aspect, what is provided is the above system, wherein the DNAbarcode comprises: (a) One or more DNA barcode modules wherein each ofthe one or more DNA barcode modules encodes information on the identityof a chemical library monomer, and (b) Optionally one or more functionalnucleic acids, and (c) Optionally, one or more nucleic acids that encodeinformation that a type of information other than information on theidentity of a chemical library monomer.

Moreover, what is provides is the above system, further comprising aplurality of caps, each capable of fitting into the opening of adifferent picowell, and each capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each capable ofminimizing or preventing leakage of fluid that is inside of thepicowell.

Also embraced is the above system, further comprising a plurality ofspherical caps, wherein each is capable of fitting into the aperture ofa picowell wherein the aperture is circular, and each capable ofminimizing or preventing evaporation of fluid that is inside of thepicowell, and each capable of minimizing or preventing leakage of fluidthat is inside of the picowell.

Also contemplated is the above system, wherein if the at least one beadcomprises a DNA barcode that takes the form of a concatenated DNAbarcode, the concatenated DNA barcode comprises: (i) A sequencing primerbinding site, (ii) A first DNA barcode module, (iii) A first annealingsite that is capable of hybridizing with a first oligonucleotide splint,wherein the first oligonucleotide splint is capable of being used toguide the enzymatic synthesis of a second DNA barcode module, (iv) Asecond DNA barcode module, (v) A second annealing site that is capableof hybridizing with a second oligonucleotide splint, wherein the secondoligonucleotide splint is capable of being used to guide the synthesisof a third DNA barcode, (vi) A third DNA barcode module, (vii) A thirdannealing site that is capable of hybridizing with a thirdoligonucleotide splint, wherein the third oligonucleotide splint iscapable of being used to synthesize a fourth DNA barcode.

In methods embodiments, what is provided is a method for screening acompound library for compounds having desired properties, comprising:(a) providing a plurality of beads, wherein each bead comprises aplurality of oligonucleotides attached to the bead surface and aplurality of substantially related compounds attached to the beadsurface, and wherein the sequence of the oligonucleotides attached tothe beads encodes the synthesis history of the plurality ofsubstantially related compounds attached to the bead surface; (b)incorporating the plurality of beads in an assay for desired propertiesof compounds in the compound library; (c) capturing a signal from atleast one bead, wherein the signal reflects the performance of thecompounds on the bead in the assay; (d) sequencing the plurality ofoligonucleotides attached to the at least one bead for which assaysignal was also captured, without removing the oligonucleotides from thebead; and (e) identifying at least one compound from the sequencingreadout of step (d) and relating it to its corresponding assayperformance captured in the signal of step (c).

In further detail, what is embraced is the above method, wherein theassay comprises a binding assay, or wherein the assay comprises anactivity assay, or wherein the assay comprises a competitive bindingassay or a competitive inhibition assay, or wherein the assay comprisesinteraction of untethered compounds with other assay reagents, whereinthe untethered compounds are compounds released from the bead surface,or wherein the compounds are released by cleaving a cleavable linkerthat connects the compounds to the beads, or wherein the assay occurs ina plurality of confined volumes, wherein nominally one bead is dispersedper confined volume.

In another aspect, what is further contemplated, is the above method,wherein the confined volume comprises an aqueous droplet, or

wherein the aqueous droplet is suspended in an oil medium or ahydrophobic liquid medium, or wherein the confined volume comprises apicowell, or wherein the picowells are organized in a regular array, orwherein the plurality of confined volumes are organized in a regulararray.

Moreover, what is further embraced is the above method, wherein theconfined volume comprises a layer of adherent aqueous medium around thebead, wherein the bead is suspended in a hydrophobic medium, and theabove method, wherein the assay reagents are washed away beforesequencing the oligonucleotides. And the above method wherein thesequencing step (d) is performed before the assay step (b). What is alsoprovided is the above method, wherein the oligonucleotides on the beadsare removed after the sequencing step, but before the assay step.Moreover, further contemplated is the above method, wherein the removingof the oligonucleotide comprises an enzymatic digestion, a chemicalcleavage, a thermal degradation or a physical shearing, and the abovemethod, wherein the binding assay comprises binding of RNA molecules tothe beads, and the above method, wherein the signal from the beadcomprises sequencing of the bound RNA molecules.

In yet another aspect, what is provided is the above method, wherein thebinding assay comprises a fluorescently labeled binding assay, whereinthe molecules binding to the compounds on the beads comprisefluorophores, or the above method, wherein the binding assay comprisesnucleic-acid labeled binding assay, wherein the molecules binding to thecompounds on the beads comprise nucleic-acid tags, wherein further thesignal from the assay comprises sequencing of the nucleic acid tagsattached to the molecules binding to the compounds on the beads.

In yet a methods embodiment relating to properties, what is provided isthe above method, wherein the desired properties include one or more of:(i) Inhibiting or stimulating the catalytic activity of an enzyme, (ii)Stimulating Th1-type immune response, as measurable by cell-based assaysor by in vivo assays, (iii) Stimulating Th2-type immune response, asmeasurable by cell-based assays or by in vivo assays, (iv) InhibitingTh1-type immune response, as measurable by cell-based assays or by invivo assays, (v) Inhibiting Th2-type immune response, as measurable bycell-based assays or by in vivo assays, (vi) Stimulating or inhibitingubiquitin-mediated degradation of a protein, as measurable by purifiedproteins, by cell-based assay, or by in vivo assays.

In a system embodiment, what is provided is a system for screening acompound library for a compound having a desired activity, comprising:(a) a sample compartment for receiving a plurality of compound-attached,oligonucleotide-encoded beads; (b) a plurality of encapsulationcompartments within the sample compartment, each encapsulationcompartment nominally comprising a single bead dispersed in an assaymedium, wherein further the assay medium comprises reagents whoseinteraction with the compounds on the beads is being assayed resultingin a measurable signal; (c) a detector for measuring signals; (d) asequencing platform; and (e) a user interface for receiving one or morecommands from a user. Also provided is the above system, wherein theencapsulation compartment comprises a liquid droplet. In another aspect,provided is the above system, wherein the encapsulation compartmentcomprises a picowell, or wherein further the encapsulation compartmentcomprises assay reagents, or wherein the detector comprises an opticaldetector, or wherein the sequencer comprises the optical detector.

In one aspect, the disclosure features a method for perturbing a cellby: (a) providing a nucleic-acid encoded perturbation and confining acell with the nucleic-acid encoded perturbation; (b) contacting the cellwith the nucleic-acid encoded perturbation in a confined volume, whereinthe perturbation initiation and dose are controlled; (c) incubating thecell with the nucleic-acid encoded perturbation for a specified periodof time; and (d) transferring the nucleic acid that encodes thenucleic-acid encoded perturbation to the cell.

In some embodiments of this aspect, the nucleic-acid encodedperturbation is a nucleic acid encoded compound or drug molecule. Insome embodiments, the nucleic-acid encoded perturbation is a DNA-encodedlibrary.

In some embodiments, the perturbation and the nucleic acid encoding theperturbation are unattached and free in solution. In some embodiments,the perturbation and the nucleic acid encoding the perturbation areattached to each other. In some embodiments, the perturbation and thenucleic acid encoding the perturbation are attached to the samesubstrate but not to each other. In some embodiments, the attachment ofthe perturbation to the substrate and the attachment of the nucleic acidto the substrate are cleavable attachments. In particular embodiments,the cleavable attachment is selected from the group consisting of aphotocleavable attachment, a temperature cleavable attachment, a pHsensitive attachment, an acid cleavable attachment, a base cleavableattachment, a sound cleavable attachment, a salt cleavable attachment, aredox sensitive attachment, or a physically cleavable attachment.

In some embodiments of this aspect of the disclosure, confining the celland the perturbation comprises a droplet encapsulation, an emulsionencapsulation, a picowell encapsulation, a macrowell encapsulation, aphysical attachment, a bubble encapsulation, or a microfluidicconfinement.

In some embodiments, the control over the perturbation comprisescontrolling light exposure, controlling temperature exposure,controlling pH exposure, controlling time exposure, controlling soundexposure, controlling salt exposure, controlling chemical or physicalredox potential, or controlling mechanical-agitation exposure.

In particular embodiments, the incubation comprises exposing the cell tothe perturbation after cleaving the perturbation from the substrate orafter cleaving the nucleic acid from the substrate. In some embodiments,the incubation comprises exposing the cell to the perturbation withoutcleaving the perturbation from the substrate or without cleaving thenucleic acid from the perturbation.

In some embodiments, transferring the nucleic acid that encodes thenucleic-acid encoded perturbation to the cell comprises attaching thenucleic acid to the cell surface of the cell. In particular embodiments,attaching the nucleic acid to the cell surface of the cell comprisesintercalating the nucleic acid into the cell membrane. In particularembodiments, attaching the nucleic acid to the cell surface of the cellcomprises attaching the nucleic acid to a biomolecule on the cellsurface. In particular embodiments, the biomolecule is a protein or acarbohydrate. In other embodiments, attaching the nucleic acid to thecell surface of the cell comprises attaching through an optional tag onthe nucleic acid.

In another aspect, the disclosure features a method for perturbing acell with a perturbation and encoding the cell with the identity of theperturbation. The method includes: (a) providing a bead-bound DNAencoded library; (b) confining a cell with the bead-bound DNA encodedlibrary, wherein the bead-bound DNA encoded library comprises one ormore copies of a combinatorially synthesized compound and one or morecopies of an encoding nucleic acid tag, wherein the compound and theencoding nucleic acid are attached to a bead, wherein the encodingnucleic acid encodes the identity of the compound, and wherein thebead-bound DNA encoded library and the cell are confined in a confiningvolume; (c) releasing the compound from the bead and incubating thecompound with the cell inside the confining volume; (d) optionallyreleasing the encoding nucleic acid tag from the bead; and (e) attachingthe encoding nucleic acid tag to the cell, thereby preserving theidentity of the compound through the encoding nucleic acid tag attachedto the cell.

In yet another aspect, the disclosure features a method for perturbing acell, encoding the cell with the identity of the perturbation, andmeasuring a response of the cell to the perturbation. The methodincludes: (a) contacting a cell with a bead-bound DNA encoded library ina first confined volume, wherein the bead-bound DNA encoded librarycomprises one or more copies of a combinatorially synthesized compoundand one or more copies of an encoding nucleic acid tag, wherein thecompound and the encoding nucleic acid are attached to a bead, andwherein the encoding nucleic acid encodes the identity of the compound;(b) releasing the compounds in the library from the bead and incubatingthe compounds in the library with the cell inside the first confinedvolume; (c) optionally releasing the encoding nucleic acid tag from thebead inside the first confined volume; (d) capturing the encodingnucleic acid tag to the cell surface of the cell, whereby the cell isexposed to the compound in the library and the identity of the compoundexposed is captured on to the cell surface; (e) releasing the cell fromthe first confining volume, wherein the encoding nucleic acid tags areattached to the cell and the encoding nucleic acid tag encodes theidentity of the compound the cell is exposed to; (f) capturing apreviously perturbed and nucleic acid tagged cell with aresponse-detection bead in a second confined volume, wherein the cell isexposed to a lysis condition that exposes the cellular content of thecell to the response-capture bead, wherein the response-capture beadcomprises capture probes that capture the cellular content and thenucleic acid tag that encodes the perturbation in the previouslyperturbed and nucleic acid tagged cell, (g) incubating theresponse-capture bead with the lysed cell in the second confiningvolume, thereby capturing both cellular content and the nucleic acid tagthat encodes the perturbation on to the response-capture bead, (h)optionally converting the response of the cell to the perturbation to anucleic acid signal, wherein the response of the cell to theperturbation is not a nucleic acid signal, and (i) sequencing thenucleic acid tag attached to the response-capture bead, therebycorrelating the identity of the perturbation to the response of the cellto the perturbation.

In still another aspect, the disclosure features a method for perturbinga cell and capturing a response of the cell to the perturbation by: (a)providing an array of picowells and a library of functionalizedperturbation beads, wherein the picowells are capable of accommodating asingle cell and a single functionalized perturbation bead, wherein eachfunctionalized perturbation bead comprises a different plurality ofsubstantially identical releasable compounds and a plurality ofnucleotide barcodes that encodes the compounds, wherein the nucleotidebarcodes are functionalized barcodes capable of capturing cellularcontent of the cell, wherein the cellular content of cell comprisescellular response to the perturbations contained in the functionalizedperturbation beads, (b) capturing single cells into each picowell of thepicowell array, (c) capturing single functionalized perturbation beadsto the picowells containing single cells, (d) releasing the compoundsfrom the functionalized perturbation beads and incubating the cells withthe released compounds, wherein the compounds between picowells haveminimal diffusion, (e) lysing the cells to release the cellularcontents, (f) capturing one or more components of the cellular contentonto functionalized oligonucleotides on the functionalized perturbationbeads, wherein the capturing comprises hybridization and enzymaticextension to combine nucleotide barcodes with nucleic acid elements ofthe cellular content, thereby forming a hybrid of the nucleotide barcodeand the nucleic acid element of the cellular content, and (g) releasingthe hybrid, collecting the hybrid from the library of functionalizedperturbation beads, and sequencing the hybrid, thereby relating theperturbation to the cellular response to the perturbation.

A system for screening chemical compounds is provided. Potentialembodiments include the following:

The system may include a picowell array plate comprising a plurality ofpicowells, each picowell has a top aperture that defines an opening atthe top of the picowell, a bottom that is defined by a floor, the topaperture is separated by a wall from the floor. The wall resides inbetween the top aperture and the floor.

The system may include a single bead disposed in a picowell, the beadcomprises a plurality of substantially identical bead bound DNAbarcodes, and a plurality of substantially identical bead boundcompounds.

The bead may comprise a bead bound DNA barcode that takes the form ofeither a concatenated DNA barcode or an orthogonal DNA barcode, and ifthe DNA barcode takes the form of a concatenated DNA barcode theconcatenated DNA barcode is made by a method that uses one or both of:uses click chemistry, or uses a repeating cycle of steps.

The repeating cycle of steps may comprise using a splint oligonucleotide(splint oligo) that is capable of hybridizing to a partially made beadbound DNA barcode. The hybridizing is mediated by an annealing site onthe splint oligo and a corresponding, complementary annealing site inthe partially made bead bound DNA barcode.

The annealed splint oligo may be used as a template for extending thepartially made DNA barcode using DNA polymerase. The splint oligocontains bases that are complementary to a DNA barcode module that is tobe polymerized to the partially made bead bound DNA barcode. The splintoligo also contains bases that are complementary to an annealing sitethat is to be polymerized to the partially made bead bound DNA barcode.

Each one of the plurality of substantially identical bead boundcompounds comprises one or more chemical library monomers, and each beadbound DNA barcode module identifies a corresponding chemical librarymonomer, the term “compound” is used to refer to a completed productthat comprises one or more chemical library members. The completed DNAbarcode identifies the compound.

The system may further comprise an oligonucleotide sequencing primerthat is capable of guiding the sequencing of one or more DNA barcodemodules that is comprised by a bead bound DNA barcode, where optionallythe system comprises a DNA sequencing machine, and where the DNAsequencing machine is not a luminescence based sequencer and not a pHbased DNA sequencing machine.

The system may further comprise a plurality of spherical caps, each capis capable of fitting into the aperture of a picowell the aperture iscircular, and each cap is capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each cap iscapable of minimizing or preventing leakage of fluid that is inside ofthe picowell.

The at least one bead disposed in the at least one picowell comprises atleast one response capture element that is coupled to said at least onebead.

The at least one of the bead disposed in a picowell comprises at leastone response capture element that is coupled to said at least one bead.The at least one response capture element comprises: Poly(dT), an exontargeting RNA probe, an antibody, or an aptamer.

The DNA barcode is either a concatenated DNA barcode or an orthogonalDNA barcode. The DNA barcode comprises one or more DNA barcode modules.Each of the one or more DNA barcode modules encodes information thatidentifies a chemical library monomer. The concatenated DNA barcode orthe orthogonal DNA barcode further includes one or both of: one or morefunctional nucleic acids, and one or more nucleic acids that encodeinformation of a type other than the identity of a chemical librarymonomer.

The bead bound concatenated DNA barcode comprises: a 1st DNA barcodemodule, or a 1st DNA barcode module, a 1st annealing site, and a 2nd DNAbarcode module, or a 1st DNA barcode module, a 1st annealing site, a 2ndDNA barcode module, a 2nd annealing site, and a 3rd DNA barcode module,or a 1st DNA barcode module, a 1st annealing site, a 2nd DNA barcodemodule, a 2nd annealing site, a 3rd DNA barcode module, a 3rd annealingsite, and a 4th DNA barcode module, or a 1st DNA barcode module, a 1stannealing site, a 2nd DNA barcode module, a 2nd annealing site, a 3rdDNA barcode module, a 3rd annealing site, a 4th DNA barcode module, a4th annealing site, and a 5th DNA barcode module, or a 1st DNA barcodemodule, a 1st annealing site, a 2nd DNA barcode module, a 2nd annealingsite, a 3rd DNA barcode module, a 3rd annealing site, a 4th DNA barcodemodule, a 4th annealing site, a 5th DNA barcode module, a 5th annealingsite, and a 6th DNA barcode module.

The bead comprises a DNA barcode that is an orthogonal DNA barcode, thebead comprises an external surface. The orthogonal DNA barcodecomprises: a first nucleic acid that comprises a first DNA barcodemodule and an annealing site for a sequencing primer, the first nucleicacid is coupled to the bead at a first position, a second nucleic acidthat comprises a second DNA barcode module and an annealing site for asequencing primer, the second nucleic acid is coupled to the bead at asecond position, and a third nucleic acid that comprises a third DNAbarcode module and an annealing site for a sequencing primer, the secondnucleic acid is coupled to the bead at a third position, and the first,second, and third position on the bead are each located at differentlocation on the bead's external surface.

The concatenated DNA barcode is made by a method that uses both clickchemistry and the repeating cycle of steps that uses the splint oligo,both click chemistry and chemical methods that are not click chemistrymethods, only click chemistry, or only the repeating cycle of steps thatuses the splint oligo.

Each of the plurality of substantially identical bead bound compounds iscoupled to the bead by way of a cleavable linker, or by way of acleavable linker that is a light cleavable linker, or by way of a noncleavable linker.

The at least one bead comprises grafted copolymers consisting of a lowcrosslinked polystyrene matrix on which polyethylene glycol (PEG) isgrafted.

At least one picowell comprises at least one cell.

The plurality of substantially identical bead bound compounds are boundto the at least one bead by way of a cleavable linker, and cleaving thecleavable linker releases the bead bound compound from the bead toproduce a released compound.

The released compound is capable of contacting the at least one cell.The at least one cell is: a mammalian cell that is not a cancer cell, amammalian cancer cell, a dead mammalian cell, an apoptotic mammaliancell, a necrotic mammalian cell, a bacterial cell, a plasmodium cell, acell that is metabolically active but has a cross linked genome and isunable to undergo cell division, or a mammalian cell that is infectedwith a virus.

Each picowell has a top aperture that defines an opening at the top ofthe picowell, a bottom that is defined by a floor, the top aperture isseparated from the floor, and a wall resides in between the top apertureand the floor, and the aperture is round. The floor is round. The walltakes the form of a truncated cone. The aperture has a first diameter,the floor has a second diameter. The first diameter is greater than thesecond diameter.

Each picowell has a top aperture that defines an opening at the top ofthe picowell, a bottom that is defined by a floor. The top aperture isseparated from the floor, and a wall resides in between the top apertureand the floor, and the aperture is round. The floor is round. The walltakes the form of a truncated cone. The aperture has a first diameter,the floor has a second diameter. The first diameter is greater than thesecond diameter.

A cap that snuggly fits into the aperture. The aperture is comprised bya polymer having a greater durometer (harder). The cap is made of apolymer having a lesser durometer (softer). The relative durometers ofthe cap and aperture allow the cap to be reversibly and snuggly fit intothe aperture.

The cap is: a cap intended only to plug the picowell and preventleakage, a cap that is a passive cap and that is capable of absorbingmetabolites that are released by a cell, in the situation where a cellin a cell medium is cultured in the picowell, a cap that is an activecap, and that takes the form of a bead that comprises a plurality ofessentially identical compounds, and each of the plurality ofessentially identical compounds is coupled to the bead with a cleavablelinker, a cap that is an active cap, and that takes the form of a beadthat comprises a plurality of identical reagents, and each of theplurality of essentially identical reagents is coupled to the bead witha cleavable linker.

The system may comprise at least one spherical cap.

The system may comprise at least one non spherical cap.

The DNA barcode comprises one or more nucleic acids that do not encodeany chemical monomer but instead identify one or more of: the class ofchemical compounds that is cleavably attached to the bead, the step in amulti step pathway of organic synthesis, a bead bound nucleic acidcorresponds to a given chemical monomer that is used to make a beadbound compound. The bead bound nucleic acid that corresponds to a givenchemical monomer identifies that chemical monomer, the date that thebead bound compound was synthesized, the disease that the bead boundcompound is intended to treat, the cellular event that the bead boundcompound is intended to stimulate or inhibit, or the reaction conditionsthat were used to couple a given chemical library monomer to the bead.

There does not exist any headpiece that links any of the bead boundcompounds to any of the bead bound DNA barcodes.

The concatenated DNA barcode comprises at least one nucleic acid that isa DNA barcode module, and at least one functional nucleic acid that: iscapable of being used as an annealing site for a sequencing primer, iscapable of forming a hairpin structure. The hairpin structure comprisesa sequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure the bend is 5 prime to the sequencingprimer and is 3 prime to the annealing site for the sequencing primer,or is a spacer nucleic acid.

The orthogonal DNA barcode contains a plurality of DNA barcode modules,each of the DNA barcode modules is coupled to a different site on thebead either directly or via a linker, and each of the plurality of DNAbarcode modules contains at least one functional nucleic acid that is:capable of being used as an annealing site for a sequencing primer,capable of forming a hairpin structure. The hairpin structure comprisesa sequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure the bend is 5 prime to the sequencingprimer and is 3 prime to the annealing site for the sequencing primer,or, a spacer nucleic acid.

A method for controlling the concentration of a compound in a solutionthat resides in a picowell. The method is applied to a bead boundcompound in a picowell. The picowell contains a solution. The bead boundcompound is coupled to the bead by way of a cleavable linker. The methodmay include the step of exposing the bead bound compound to a conditionthat effects cleavage of the cleavable linker. The condition compriseslight that is capable of cleaving the cleavable linker.

The method may include the step of allowing release of the bead boundcompound from the bead to generate a released compound, release isfollowed by diffusion or dispersion of the released compound in thesolution to result in a substantially uniform concentration of thecompound in the solution.

The method may include the step of adjusting the condition to produce adetermined concentration of the substantially uniform concentration. Thedetermined concentration is made with regard to the concentration of areleased fluorophore that is released by from a bead bound releasemonitor.

The condition is adjusted by adjusting one or more of the wavelength ofthe light, the intensity of the light, and by the duration of lightexposure and, optionally: the concentration of a released fluorophorethat is released from a bead bound release monitor is determined at thesame time as effecting release of the bead bound compound from the beadto generate a released compound, or the concentration of a releasedfluorophore that is released from a bead bound release monitor isdetermined at a time substantially before effecting release of the beadbound compound from the bead to generate a released compound.

A cap in combination with a picowell plate that comprises a plurality ofpicowells. The cap is capable of use with said picowell plate.

Each of the plurality of picowells is definable by an aperture, a floor,and a wall. The wall is defined by the aperture on top and the floor onthe bottom. The aperture is round. The floor is round. The wall takesthe form of a surface of a truncated cone, and the aperture has a firstdiameter, the floor has a second diameter. The first diameter is greaterthan the second diameter. The cap is a spherical cap that is capable ofsnuggly fitting into the aperture. The aperture is comprised by apolymer having a greater durometer (harder). The cap is made of apolymer having a lesser durometer (softer). The relative durometers ofthe cap and aperture allow the spherical cap to be reversibly andsnuggly fit into the aperture. The cap is: capable of plugging thepicowell and preventing leakage, a passive cap and that is capable ofabsorbing metabolites that are released by a cell, in the situationwhere a cell in a cell medium is cultured in the picowell, an active capthat takes the form of a bead that comprises a plurality of essentiallyidentical compounds, and each of the plurality of essentially identicalcompounds is coupled to the bead with a cleavable linker, at least oneof the plurality of picowells contains an aqueous medium, and cleavageof the cleavable linker releases at least some of the plurality ofessentially identical compounds from the bead into the aqueous medium.

A system comprising a picowell array plate comprising an upper generallyplaner surface, a plurality of picowells, each picowell has a topaperture that defines an opening at the top of the picowell, a bottomthat is defined by a floor. The top aperture is separated by a wall fromthe floor. The wall resides in between the top aperture and the floor,and optionally, a bead disposed in at least one of said plurality ofpicowells. The bead comprises a plurality of substantially identicalbead bound DNA barcodes, and a plurality of substantially identical beadbound compounds, the picowell array plate further comprises a mat thatis capable of securely covering the opening at the top of at least oneor all of the plurality of picowells, or that is actually securelycovering the opening at the top of at least one or all of the pluralityof picowells. The securely covering is reversible. The mat optionallycomprises one or all of: an absorbent surface that, when positioned incontact with the upper generally planer surface of the picowell arrayplate, is capable of absorbing any metabolites, biochemicals, orproteins that may be comprised by one or more of the plurality ofpicowells, an adhesive surface that is capable of maintaining reversibleadhesion to the top generally planer surface of the picowell arrayplate.

A method for determining a signal from an assay and a sequencing readouton a bead, thereby identifying one or more compounds of interest fromthe assay, comprising the steps: providing a plurality of beads, eachbead comprises a plurality of compounds attached to the bead that arerelated substantially to each other, and a plurality ofoligonucleotides. The plurality of oligonucleotides attached to eachbead identify the plurality of compounds attached to the same bead,performing the assay involving the plurality of compounds attached tothe beads, determining at least one signal that reflects the performanceof the compounds in the assay of step b, sequencing the plurality ofoligonucleotides attached to the beads, without removing theoligonucleotides from the bead, thereby determining a sequencing readoutfor each bead, and identifying the compounds attached to the bead by thesequencing readout of step d and relating it to the assay performancecontained in the determined signal of step c, beads having a signal fromthe assay and the sequencing readout identify the compound of interest.

A method for screening a compound library for compounds having desiredproperties, comprising: providing a plurality of beads, each beadcomprises a plurality of oligonucleotides attached to the bead surfaceand a plurality of substantially related compounds attached to the beadsurface. The sequence of the oligonucleotides attached to the beadsencodes the identity of the plurality of substantially related compoundsattached to the bead surface, incorporating the plurality of beads in anassay for desired properties of compounds in the compound library,capturing a signal from at least one bead. The signal reflects theperformance of the compounds on the bead in the assay, sequencing theplurality of oligonucleotides attached to the at least one bead forwhich assay signal was also captured, without removing theoligonucleotides from the bead, and identifying at least one compoundfrom the sequencing readout of step (d) and relating it to itscorresponding assay performance captured in the signal of step (c).

Each bead comprises a different plurality of oligonucleotides and adifferent plurality of substantially related compounds.

The plurality of oligonucleotides is a plurality of DNAoligonucleotides.

The plurality of compounds is attached to the bead surface by joiningmultiple compound building blocks in tandem, all the compound buildingblocks together make up the compound.

Each DNA module and each compound building block are assembledsequentially and alternatively.

Each compound in the plurality of identical compounds is attached to thebead surface by way of a cleavable linker.

The cleavable linker is a photocleavable linker, a protease cleavablelinker, or an acid cleavable linker.

The compounds are cleaved from the bead surface after step (a) and priorto step (d).

The signal that reflects the desired property of the compound is afluorescent signal.

The size of each bead is between 1 μm and 100 μm.

The size of each bead is between 1 μm and 10 μm.

The size of each bead is about 3 μm.

The method further comprises identifying a target candidate in aplurality of potential targets. The compound having the desired propertybinds to the target candidate.

The step (b) comprises incubating the plurality of beads in theplurality of potential targets.

The potential targets are proteins or nucleic acids.

The sequencing is performed by single-molecule real-time sequencing, ionsemiconductor sequencing, pyrosequencing, sequencing by synthesis,sequencing by bridge amplification, sequencing by ligation, nanoporesequencing, chain termination sequencing, massively parallel signaturesequencing, polony sequencing, heliscope single molecule sequencing,shotgun sequencing, SOLiD sequencing, Illumina sequencing, tunnelingcurrents DNA sequencing, sequencing by hybridization, sequencing withmass spectrometry, microfluidic Sanger sequencing, and oligonucleotideextension sequencing.

A method for screening a compound library for compounds having desiredproperties, comprising: providing a plurality of beads, each beadcomprises a plurality of oligonucleotides attached to the bead surfaceand a plurality of substantially related compounds attached to the beadsurface. The sequence of the oligonucleotides attached to the beadsencodes the synthesis history of the plurality of substantially relatedcompounds attached to the bead surface, incorporating the plurality ofbeads in an assay for desired properties of compounds in the compoundlibrary, capturing a signal from at least one bead. The signal reflectsthe performance of the compounds on the bead in the assay, sequencingthe plurality of oligonucleotides attached to the at least one bead forwhich assay signal was also captured, without removing theoligonucleotides from the bead, and identifying at least one compoundfrom the sequencing readout of step (d) and relating it to itscorresponding assay performance captured in the signal of step (c).

The assay comprises a binding assay.

The assay comprises an activity assay.

The assay comprises a competitive binding assay or a competitiveinhibition assay.

The assay comprises interaction of untethered compounds with other assayreagents. The untethered compounds are compounds released from the beadsurface.

The compounds are released by cleaving a cleavable linker that connectsthe compounds to the beads.

The assay occurs in a plurality of confined volumes, nominally one beadis dispersed per confined volume.

The confined volume comprises an aqueous droplet.

The aqueous droplet is suspended in an oil medium or a hydrophobicliquid medium.

The confined volume comprises a picowell.

The picowells are organized in a regular array.

The plurality of confined volumes are organized in a regular array.

The confined volume comprises a layer of adherent aqueous medium aroundthe bead. The bead is suspended in a hydrophobic medium.

The assay reagents are washed away before sequencing theoligonucleotides.

The sequencing step (d) is performed before the assay step (b).

The oligonucleotides on the beads are removed after the sequencing step,but before the assay step.

The removing of the oligonucleotide comprises an enzymatic digestion, achemical cleavage, a thermal degradation or a physical shearing.

The binding assay comprises binding of RNA molecules to the beads.

The signal from the bead comprises sequencing of the bound RNAmolecules.

The binding assay comprises a fluorescently labeled binding assay. Themolecules binding to the compounds on the beads comprise fluorophores.

The binding assay comprises nucleic-acid labeled binding assay. Themolecules binding to the compounds on the beads comprise nucleic-acidtags, further the signal from the assay comprises sequencing of thenucleic acid tags attached to the molecules binding to the compounds onthe beads.

The desired properties include one or more of: inhibiting or stimulatingthe catalytic activity of an enzyme, stimulating Th1 type immuneresponse, as measurable by cell based assays or by in vivo assays,stimulating Th2 type immune response, as measurable by cell based assaysor by in vivo assays, inhibiting Th1 type immune response, as measurableby cell based assays or by in vivo assays, inhibiting Th2 type immuneresponse, as measurable by cell based assays or by in vivo assays,stimulating or inhibiting ubiquitin mediated degradation of a protein,as measurable by purified proteins, by cell based assay, or by in vivoassays.

A system for screening a compound library for a compound having adesired activity, comprising: a sample compartment for receiving aplurality of compound-attached, oligonucleotide-encoded beads, aplurality of encapsulation compartments within the sample compartment,each encapsulation compartment nominally comprising a single beaddispersed in an assay medium, further the assay medium comprisesreagents whose interaction with the compounds on the beads is beingassayed resulting in a measurable signal, a detector for measuringsignals, a sequencing platform, and a user interface for receiving oneor more commands from a user.

The encapsulation compartment comprises a liquid droplet.

The encapsulation compartment comprises a picowell.

The encapsulation compartment comprises assay reagents.

The detector comprises an optical detector.

The sequencer comprises the optical detector.

A method for perturbing a cell, comprising: providing a nucleic-acidencoded perturbation and confining a cell with the nucleic-acid encodedperturbation, contacting the cell with the nucleic-acid encodedperturbation in a confined volume. The perturbation initiation and doseare controlled, incubating the cell with the nucleic-acid encodedperturbation for a specified period of time, and transferring thenucleic acid that encodes the nucleic-acid encoded perturbation to thecell.

The nucleic-acid encoded perturbation is a nucleic acid encoded compoundor drug molecule.

The nucleic-acid encoded perturbation is a DNA-encoded library.

The perturbation and the nucleic acid encoding the perturbation areunattached and free in solution.

The perturbation and the nucleic acid encoding the perturbation areattached to each other.

The perturbation and the nucleic acid encoding the perturbation areattached to the same substrate but not to each other.

The attachment of the perturbation to the substrate and the attachmentof the nucleic acid to the substrate are cleavable attachments.

The cleavable attachment is selected from the group consisting of aphotocleavable attachment, a temperature cleavable attachment, a pHsensitive attachment, an acid cleavable attachment, a base cleavableattachment, a sound cleavable attachment, a salt cleavable attachment, aredox sensitive attachment, or a physically cleavable attachment.

Confining the cell and the perturbation comprises a dropletencapsulation, an emulsion encapsulation, a picowell encapsulation, amacrowell encapsulation, a physical attachment, a bubble encapsulation,or a microfluidic confinement.

The control over the perturbation comprises controlling light exposure,controlling temperature exposure, controlling pH exposure, controllingtime exposure, controlling sound exposure, controlling salt exposure,controlling chemical or physical redox potential, or controllingmechanical-agitation exposure.

The incubation comprises exposing the cell to the perturbation aftercleaving the perturbation from the substrate or after cleaving thenucleic acid from the substrate.

The incubation comprises exposing the cell to the perturbation withoutcleaving the perturbation from the substrate or without cleaving thenucleic acid from the perturbation.

Transferring the nucleic acid that encodes the nucleic-acid encodedperturbation to the cell comprises attaching the nucleic acid to thecell surface of the cell.

Attaching the nucleic acid to the cell surface of the cell comprisesintercalating the nucleic acid into the cell membrane.

Attaching the nucleic acid to the cell surface of the cell comprisesattaching the nucleic acid to a biomolecule on the cell surface.

The biomolecule is a protein or a carbohydrate.

Attaching the nucleic acid to the cell surface of the cell comprisesattaching through an optional tag on the nucleic acid.

A method for perturbing a cell with a perturbation and encoding the cellwith the identity of the perturbation, comprising: providing abead-bound DNA encoded library, confining a cell with the bead-bound DNAencoded library. The bead-bound DNA encoded library comprises one ormore copies of a combinatorially synthesized compound and one or morecopies of an encoding nucleic acid tag. The compound and the encodingnucleic acid are attached to a bead. The encoding nucleic acid encodesthe identity of the compound. The bead-bound DNA encoded library and thecell are confined in a confining volume, releasing the compound from thebead and incubating the compound with the cell inside the confiningvolume, optionally releasing the encoding nucleic acid tag from thebead, and attaching the encoding nucleic acid tag to the cell, therebypreserving the identity of the compound through the encoding nucleicacid tag attached to the cell.

A method for perturbing a cell, encoding the cell with the identity ofthe perturbation, and measuring a response of the cell to theperturbation, comprising: contacting a cell with a bead-bound DNAencoded library in a first confined volume. The bead-bound DNA encodedlibrary comprises one or more copies of a combinatorially synthesizedcompound and one or more copies of an encoding nucleic acid tag. Thecompound and the encoding nucleic acid are attached to a bead. Theencoding nucleic acid encodes the identity of the compound, releasingthe compounds in the library from the bead and incubating the compoundsin the library with the cell inside the first confined volume,optionally releasing the encoding nucleic acid tag from the bead insidethe first confined volume, capturing the encoding nucleic acid tag tothe cell surface of the cell, whereby the cell is exposed to thecompound in the library and the identity of the compound exposed iscaptured on to the cell surface, releasing the cell from the firstconfining volume. The encoding nucleic acid tags are attached to thecell and the encoding nucleic acid tag encodes the identity of thecompound the cell is exposed to, capturing a previously perturbed andnucleic acid tagged cell with a response-detection bead in a secondconfined volume. The cell is exposed to a lysis condition that exposesthe cellular content of the cell to the response-capture bead. Theresponse capture bead comprises capture probes that capture the cellularcontent and the nucleic acid tag that encodes the perturbation in thepreviously perturbed and nucleic acid tagged cell, incubating theresponse-capture bead with the lysed cell in the second confiningvolume, thereby capturing both cellular content and the nucleic acid tagthat encodes the perturbation on to the response-capture bead,optionally converting the response of the cell to the perturbation to anucleic acid signal. The response of the cell to the perturbation is nota nucleic acid signal, and sequencing the nucleic acid tag attached tothe response-capture bead, thereby correlating the identity of theperturbation to the response of the cell to the perturbation.

A method for perturbing a cell and capturing a response of the cell tothe perturbation, comprising: providing an array of picowells and alibrary of functionalized perturbation beads. The picowells are capableof accommodating a single cell and a single functionalized perturbationbead, each functionalized perturbation bead comprises a differentplurality of substantially identical releasable compounds and aplurality of nucleotide barcodes that encodes the compounds. Thenucleotide barcodes are functionalized barcodes capable of capturingcellular content of the cell. The cellular content of cell comprisescellular response to the perturbations contained in the functionalizedperturbation beads, capturing single cells into each picowell of thepicowell array, capturing single functionalized perturbation beads tothe picowells containing single cells, releasing the compounds from thefunctionalized perturbation beads and incubating the cells with thereleased compounds. The compounds between picowells have minimaldiffusion, lysing the cells to release the cellular contents, capturingone or more components of the cellular content onto functionalizedoligonucleotides on the functionalized perturbation beads. The capturingcomprises hybridization and enzymatic extension to combine nucleotidebarcodes with nucleic acid elements of the cellular content, therebyforming a hybrid of the nucleotide barcode and the nucleic acid elementof the cellular content, and releasing the hybrid, collecting the hybridfrom the library of functionalized perturbation beads, and sequencingthe hybrid, thereby relating the perturbation to the cellular responseto the perturbation.

A system for screening chemical compounds for their ability to modulatethe biological activity of a cell or a component of a cell is provided.The system may include an assay device comprising a multiplicity ofwells wherein each well is separated from other wells. The system mayinclude a plurality of beads where a single bead is disposed in eachwell. Each bead may comprise a plurality of substantially identicalbead-bound compounds. The bead-bound compounds may be covalently linkedto the bead by a cleavable linker such that said compounds may bereleasable from said bead in a measurable dose dependent manner as partof an assay.

Also, a method for screening chemical compounds for their ability tomodulate the biological activity of a cell or a component of a cell isprovided. With the method, a system may be provided. The system mayinclude an assay device comprising a multiplicity of wells each well maybe separated from other wells. The system may include a plurality ofbeads where each bead may be suitable for disposal in each well, eachbead may comprise a plurality of substantially identical bead-boundcompounds, said bead-bound compounds may be covalently linked to thebead by a cleavable linker such that said compounds may be releasablefrom said bead in a measurable dose dependent manner as part of anassay.

The beads may further comprise a plurality of substantially identicalbead-bound DNA barcodes linked to the bead (i) by a cleavable linker or(ii) by a non-cleavable linker, if the DNA barcodes may be linked to thebead by a cleavable linker, the cleavable linker may be orthogonal tothe cleavable linker used to link the bead-bound compounds to the bead,and the DNA barcode identifies the compound.

The system further includes transferring beads into the wells using atransfer dispenser that permits only a single bead to be included in asingle well. In one embodiment, these devices have a multiplicity ofcavities each cavity may have a single opening diameter and may beconfigured to hold only a single bead of the plurality of beads. Theassay device and the transfer dispenser may be configured to fit onto ormate with each other, optionally with a gap being present between saidassay device and said transfer device, and the cavities of the transferdispenser may be aligned with the wells of the device when the assaydevice and the transfer dispenser may be fitted onto or mated with eachother. Transfer dispensers are further described in International PatentApplication No. PCT/US2022/032559, titled “Transfer Dispensers for AssayDevices with Bead Size Exclusion,” filed on Jun. 7, 2022, which isherein incorporated by reference in its entirety.

Each well may have an opening diameter that may be larger than theopening diameter of each of said cavities such that, when each cavitymay be placed over each well, a containment space may be formedcomprising said cavity and said well.

The single bead may be released from the cavity through said containmentspace and deposited into said well.

The beads may be non-magnetic beads.

Said gap when present may have a size (e.g., diameter) that may besmaller than a size (e.g., diameter) of the bead being transferredwithin the device. This prevents movement of the beads through the gap.

With the method, a device may be provided. The device may have at leastone processor and a memory storing at least one program for execution bythe at least one processor, the at least one program includinginstructions, which, when executed by the at least one processor causethe at least one processor to perform operations.

The operations may include moving the assay device and the transferdispenser to fit onto or mate with each other, optionally with a gapbeing present between said assay device and said transfer device.

The operations may include aligning the cavities of the transferdispenser with the wells of the device.

The operations may include fitting onto or mating the assay device andthe transfer dispenser with each other to form a containment space.

The operations may include releasing the single bead from the cavitythrough said containment space.

Furthermore, a non-transitory computer-readable storage medium storingat least one program for screening chemical compounds for their abilityto modulate the biological activity of a cell or a component of a cellis provided. With the non-transitory computer-readable storage medium, asystem may be provided. The system may include an assay devicecomprising a multiplicity of wells each well may be separated from otherwells. The system may include a plurality of beads where each bead maybe suitable for disposal in each well, each bead may comprise aplurality of substantially identical bead-bound compounds, saidbead-bound compounds may be covalently linked to the bead by a cleavablelinker such that said compounds may be releasable from said bead in ameasurable dose dependent manner as part of an assay.

Said beads may further comprise a plurality of substantially identicalbead-bound DNA barcodes linked to the bead (i) by a cleavable linker or(ii) by a non-cleavable linker, if the DNA barcodes may be linked to thebead by a cleavable linker, the cleavable linker may be orthogonal tothe cleavable linker used to link the bead-bound compounds to the bead,and the DNA barcode identifies the compound.

With the non-transitory computer-readable storage medium, the system mayfurther include a transfer dispenser having a multiplicity of cavitieseach cavity may have a single opening diameter and may be configured tohold only a single bead of the plurality of beads. The assay device andthe transfer dispenser may be configured to fit onto or mate with eachother, optionally with a gap being present between said assay device andsaid transfer device, and the cavities of the transfer dispenser may bealigned with the wells of the device when the assay device and thetransfer dispenser may be fitted onto or mated with each other.

Each well may have an opening diameter that may be larger than theopening diameter of each of said cavities such that, when each cavitymay be placed over each well, a containment space may be formedcomprising said cavity and said well.

The single bead may be released from the cavity through said containmentspace and deposited into said well.

The beads may be non-magnetic beads.

Said gap when present may have a size (e.g., diameter) that may besmaller than a size (e.g., diameter) of the bead being transferredwithin the device.

The at least one program may be for execution by at least one processorand a memory storing the at least one program, the at least one programincluding instructions, when, executed by the at least one processorcause the at least one processor to perform operations. The operationsmay include moving the assay device and the transfer dispenser to fitonto or mate with each other, optionally with a gap being presentbetween said assay device and said transfer device. The operations mayinclude moving aligning the cavities of the transfer dispenser with thewells of the device. The operations may include moving fitting onto ormating the assay device and the transfer dispenser with each other toform a containment space. The operations may include moving releasingthe single bead from the cavity through said containment space. Theoperations may include moving depositing the single bead into said well.

The beads may further comprise a plurality of substantially identicalbead-bound DNA barcodes linked to the bead (i) by a cleavable linker or(ii) by a non-cleavable linker. If the DNA barcodes are linked to thebead by a cleavable linker, then the cleavable linker may be orthogonalto the cleavable linker used to link the bead-bound compounds to thebead, and the DNA barcode may identify the compound.

The system may further include a transfer dispenser having amultiplicity of cavities each cavity has a single opening diameter andis configured to hold only a single bead of the plurality of beads. Theassay device and the transfer dispenser may be configured to fit onto ormate with each other, optionally with a gap being present between saidassay device and said transfer device. The cavities of the transferdispenser may be aligned with the wells of the device when the assaydevice and the transfer dispenser are fitted onto or mated with eachother.

Each well may have an opening diameter that is larger than the openingdiameter of each of said cavities such that. When each cavity is placedover each well, a containment space may be formed comprising said cavityand said well.

The single bead may be released from the cavity through said containmentspace and deposited into said well.

The beads may be non-magnetic beads.

The gap when present may have a size (e.g., diameter) that may besmaller than a size (e.g., diameter) of the bead being transferredwithin the device.

Each of the above-referenced system, method and non-transitorycomputer-readable storage medium may include one or more of each of thefeatures listed below in any suitable combination.

When the bead-bound compounds are released from the bead, the bead-boundDNA barcodes may be not released from the bead.

Each of the confined volumes may comprise a single bead.

The confined volume may be either a picowell or a droplet.

The cleavable covalent linker may be cleaved by light, temperaturechange, pH change, sound, salt or a change in oxidation.

The cleavable linker may be cleaved by light.

The light may be ultraviolet light.

The component of a cell may be a lipid, a protein, a carbohydrate, or anucleic acid.

The component of a cell may be a nucleic acid.

The nucleic acid may be mRNA.

The component of a cell may be a cytokine, an antigen, or an enzyme.

The amount of compound released from the bead in a measurable dosedependent manner for testing may be measured by a change in signalgenerated from a signal generating compound bound to a bead and distinctfrom the compound to be tested, the signal generating compound generatesno signal or an attenuated signal on the bead and may be capable ofgenerating an increased signal once released from the bead, the increasein signal correlates to the amount of the to be tested compound releasedfrom the bead, the compound released from the bead and the signalgenerating compound released from the bead may be released under thesame conditions.

The compound and the signal generating compound may be bound to the samebead.

The signal generating compound may be a fluorescent compound whosefluorescence may be quenched by a quencher when both may be bond to thebead.

At least one confined volume may comprise a bead with bead-bound DNAbarcodes and bead-bound compounds that may be different from thebead-bound DNA barcodes and bead-bound compounds on a bead in anotherconfined volume.

The DNA barcode may be linked to a bead by a cleavable linker and thecleavable linker may be different from the cleavable linker linking thebead-bound compound to the bead.

The DNA barcode may be linked to the bead by a non-cleavable linker.

The DNA barcode may be linked to the bead by a cleavable linker and thecleavable linker may be cleaved by a different mechanism than thecleavable linker linking the bead-bound compound to the bead.

The transfer dispenser further may comprise a locking mechanism thatallows the transfer dispenser and the assay device to be locked intoproper alignment.

A diameter of the each of the cavities may be reversibly expandable toaccommodate the bead.

A diameter of each of the cavities may be at least 100.1% of thediameter of the bead, either in the native or expanded form toaccommodate the bead.

A depth of each of the cavities may be at least 50% of the size (e.g.,diameter) of the bead up to about 125% of the size (e.g., diameter) ofthe bead.

The transfer dispenser may be configured to integrate with a containmentcap for the delivery of the bead to the multiplicity of cavities of thetransfer dispenser under the assay device.

The single bead may be a size excluded bead such that the size (e.g.,diameter) of said bead excludes beads having a size (e.g., diameter)that result in two beads fitting into a single cavity.

The beads may have a size (e.g., diameter) of from about 0.5 to about100 microns.

The transfer dispenser may be configured to may have fitted thereon saidassay device, and said transfer dispenser may be positioned below andaligned to the assay device in an inverted position such that eachcavity in the transfer dispenser may be aligned to a single well in theassay device provided that said transfer dispenser contains no more thana single bead in a given cavity.

The transfer dispenser may be configured to may have fitted thereon saidassay device, and said assay device may be positioned below and alignedto the transfer dispenser such that each well in the assay device maycomprise a single bead, which may be transferred from said transferdispenser, and said transfer requires a single fitting of the assaydevice to the transfer dispenser.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise a bottom surface opposite an open end of the cavity, thebottom surface may comprise a sub-cavity recessed into the bottomsurface, and the sub-cavity may be configured to hold the single bead.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise an open bottom surface and an open top surface, a size ofthe open bottom surface may be smaller than a size of the open topsurface, and the cavity may be configured so that the single bead comesto rest at an intermediate point between the open top surface and theopen bottom surface.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise a bottom surface opposite an open end of the cavity, thebottom surface may comprise a magnet in or on the bottom surface, andthe magnet may be configured to hold the single bead.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise an opening with an inner size, and the opening may beblocked with a stop structure, and the stop structure may comprise atleast one of the group consisting of a mesh of solid material withsub-openings therein, a porous fabric, and a sub-structure having atleast one sub-opening having a size smaller than the inner size of theopening and larger than a maximum dimension of the single bead.

A system for screening chemical compounds for their ability to modulatethe biological activity of a cell or a component of a cell is provided.The system may include an assay device comprising a multiplicity ofwells wherein each well is separated from other wells, wherein themultiplicity of wells includes at least 50,000 wells. The system mayinclude a plurality of beads where each bead is suitable for disposal ineach well. Each bead may comprise a plurality of substantially identicalbead-bound compounds. Said bead-bound compounds may be covalently linkedto the bead by a cleavable linker such that said compounds arereleasable from said bead in a measurable dose dependent manner as partof an assay.

In other embodiments, the system may include an assay device comprisinga multiplicity of wells wherein each well is separated from other wells,a plurality of beads where a single bead is suitable for disposal in asingle well, wherein each bead comprises a plurality of substantiallyidentical bead-bound compounds, wherein the bead-bound compounds arecovalently linked to the bead by a cleavable linker such that thecompounds are releasable from the bead in a measurable dose dependentmanner as part of an assay, the beads further including a plurality ofsubstantially identical bead-bound DNA barcodes linked to the bead (i)by a cleavable linker or (ii) by a non-cleavable linker, wherein if theDNA barcodes are linked to the bead by a cleavable linker, the cleavablelinker is orthogonal to the cleavable linker used to link the bead-boundcompounds to the bead, and wherein the DNA barcode identifies thecompound; and wherein each bead includes at least about 10,000substantially identical DNA barcodes.

Said beads may further comprise a plurality of substantially identicalbead-bound DNA barcodes linked to the bead (i) by a cleavable linker or(ii) by a non-cleavable linker. If the DNA barcodes are linked to thebead by a cleavable linker, the cleavable linker may be orthogonal tothe cleavable linker used to link the bead-bound compounds to the bead.The DNA barcode may identify the compound.

The system may further comprise a transfer dispenser capable ofdispensing a single bead into a single well.

The transfer device may further comprise at least one pipette capable oftransmitting the single bead, the at least one pipette including aflexible tip. The flexible tip of the pipette may include polyimide. Theflexible tip may extend along no more than 20% of a total length of thepipette. The flexible tip may extend along no more than 10% of a totallength of the pipette.

A method for screening chemical compounds for their ability to modulatethe biological activity of a cell or a component of a cell is provided.The method may be performed with the above-referenced system.

The method may comprise moving the assay device and the transferdispenser to fit onto or mate with each other, optionally with a gapbeing present between said assay device and said transfer device. Themethod may comprise aligning the cavities of the transfer dispenser withthe wells of the device. The method may comprise fitting onto or matingthe assay device and the transfer dispenser with each other to form acontainment space. The method may comprise releasing the single beadfrom the cavity through said containment space. The method may comprisedepositing the single bead into said well.

The transfer device may be operated robotically, or manually or by acombination of robotic and manual processes.

The transfer device may employ magnetic attraction, electrostaticattraction or engineering principles based on size and gravity todeposit a single bead into a single well.

The transfer dispenser may comprise a multiplicity of cavities.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise a bottom surface opposite an open end of the cavity. Thebottom surface may comprise a sub-cavity recessed into the bottomsurface. The sub-cavity may be configured to hold the single bead.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise an open bottom surface and an open top surface. A size ofthe open bottom surface may be smaller than a size of the open topsurface. The cavity may be configured so that the single bead comes torest at an intermediate point between the open top surface and the openbottom surface.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise a bottom surface opposite an open end of the cavity. Thebottom surface may comprise a magnet in or on the bottom surface. Themagnet may be configured to hold the single bead.

Each cavity of the multiplicity of cavities of the transfer dispensermay comprise an opening with an inner size. The opening may be blockedwith a stop structure. The stop structure may comprise at least one ofthe group consisting of a mesh of solid material with sub-openingstherein, a porous fabric, and a sub-structure having at least onesub-opening having a size smaller than the inner size of the opening andlarger than a maximum dimension of the single bead.

A method for identifying a transcriptome change in a cell induced by acompound, wherein said compound is included in an assay of acombinatorial library. The method may be performed with theabove-referenced system.

The method may include generating an assay array. The assay array mayinclude a plurality of wells wherein each well is separated from otherwells and each well comprises at least one cell of interest whereinassay array comprises over 50,000 wells. The assay array may include aplurality of beads where a single bead wherein each bead comprises aplurality of same bead-bound compound such that each bead comprises aunique compound from said combinatorial library and each compound insaid library is selected as a potential drug candidate. The assay arraymay include a plurality of functionalized oligonucleotides. Thefunctionalized oligonucleotide may comprise an oligonucleotide portionthat encodes the structure of the unique compound or the synthetic stepsused to make said unique compound and a RNA capturing element. A singlebead may disposed in a single well.

The method may include contacting the cell in each confined volume withthe compound released into the confined volume from the bead andmaintaining said contact for a period sufficient to generate atranscriptome change in the RNA expressed by the cell in response to thesaid contacting.

The method may include capturing RNA from the cell in each well bylysing the cell and contacting the RNA with the RNA capturing element onsaid bead.

The method may include identifying the captured RNA from at least aportion of the plurality of beads and assessing any transcriptome changein said captured RNA.

The method may include identifying the structure of the compound thatgenerated said transcriptome change.

The beads may be added to the wells of said assay array using a transferdevice capable of dispensing a single bead into a single well.

These and other capabilities of the disclosed subject matter will bemore fully understood after a review of the following figures, detaileddescription, and claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

These and other features will be more readily understood from thefollowing detailed description taken in conjunction with theaccompanying drawings, in which:

FIG. 1 . Concatenated-style bead. In concatenated-style bead, the DNAbarcode takes the form of all of the DNA barcode modules connected toeach other in a single chain, together with any other nucleic acids thathave functions, such as primer annealing sites, as a spacer, orinformation on date of manufacture. The numbers on this figure are notstructure numbers. The numbers refer to the sequence of “DNA barcodemodules” in the DNA barcode.

FIG. 2 . Orthogonal-style bead. In orthogonal-style bead, the DNAbarcode takes the form of all of the DNA barcode modules, where the DNAbarcode modules do not occur together in a single chain, but insteadoccur separately linked to different positions on the bead. The numberson this figure are not structure numbers. The numbers refer to thesequence of “DNA barcode modules” in the DNA barcode.

FIG. 3 . Cleavable linkers, conditions for cleavage (UV light orchemical), and cleavage products. Information from, Yinliang Yang (2014)Design of Cleavable Linkers and Applications in Chemical Proteomics.Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. Thealphabet letter to the left of each linker is from this reference.

FIG. 4 . Exemplary amino acid derivatives for the compositions andmethods of the present disclosure.

FIG. 5 . The photograph discloses increases in degradation of a fusionprotein, inside HeLa cells, with increasing concentrations of addedlenalidomide. Top: Expression of IKZF1/GFP fusion protein. Bottom:Expression of mScarlett® control. Lenalidomide was added at zero, 0.1,1.0, or 10 micromolar.

FIG. 6 . The photograph discloses increases in degradation of a fusionprotein, inside HeLa cells, with increasing concentrations of addedlenalidomide. Top: Expression of IKZF3/GFP fusion protein. Bottom:Expression of mScarlett control. Lenalidomide was added at zero, 0.1,1.0, or 10 micromolar.

FIG. 7 . Methods and reagents for creating bead-bound DNA barcode. Themost accurate description of “DNA barcode” is the sum of all of theinformation that is contained in the sum of all DNA barcode modules. Butfor convenience, the term “DNA barcode” is used herein to refer to thesum of all of the information of all of the DNA barcode modules plus anyadditional nucleic acids that provide information such as step number,or general type of chemical monomers that make up the bead-boundcompound, and plus any additional nucleic acids that serve a function,such as linker, sequencing primer binding site, hairpin with sequencingprimer binding site, or spacer. Where a DNA barcode is made, at least inpart, by way of click chemistry, the DNA barcode may include residualchemical groups from the click chemistry reactions.

FIG. 8 . Structure of Alexa Fluor® 488. A goal of this figure is toidentify the compound without having to resort to using the trade name.

FIG. 9 . Simplified diagram of bead-bound release-monitor. Therelease-monitor provides the user with a measure of the concentration ofthe soluble compound, following UV-induced release of the compound fromthe bead. In a preferred embodiment, one type of bead is dedicated tobeing a release-monitor, that is, this bead does not also containbead-bound compound and does not also contain bead-bound DNA library.“PCL” is photocleavable linker.

FIG. 10 . Detailed diagram of bead release-monitor.

FIG. 11 . Chemical synthesis of bead release-monitor.

FIG. 12 . Amine-functionalized bead with bifunctional linker, where thelinker includes a lysine residue.

FIG. 13 . Steps of chemical synthesis of lenalidomide modified with afirst type of carboxyl group.

FIG. 14 . Steps of chemical synthesis of lenalidomide modified with asecond type of carboxyl group.

FIG. 15 . Steps of chemical synthesis of lenalidomide modified with athird type of carboxyl group.

FIG. 16A, FIG. 16B, FIG. 16C. Lenalidomide analogues.

FIG. 17 . Steps of chemical synthesis of a deoxycytidine analoguesuitable for click-chemistry synthesis of a DNA barcode.

FIGS. 18A,B,C. Caps for placing over the top of picowells and forsealing the picowells. FIG. 18A shows active cap, where compound isreleasable by way of cleavable linker. FIG. 18B shows another type ofactive cap, where a reagent such as an antibody is bound. The boundreagent can be permanently linked, it can be linked by a cleavablelinker, or it can be bound by way of hydrogen bonds and be releasablemerely by exposure to the solution in the picowell followed by diffusionaway from the active cap and into this solution.

FIG. 18C shows a passive cap, which can be used to absorb, adsorb,collect, or capture metabolites from the solution in the picowell. Theabsorbed metabolites can subsequently be analyzed.

FIGS. 19A,B,C. FIG. 19A Picowell plate without caps over the picowells.FIG. 19B. Picowell plate with a cap over each picowell. FIG. 19C.Polyacrylamide solution being poured over the picowell plate that hasone cap securely fastened over each picowell. The polyacrylamide thenseeps into the porous cap, solidifies, and forms a stable adhesion toeach cap. FIG. 19D. The solidified polyacrylamide “roof” is then peeledoff from the picowell plate, bringing with it each cap. The metabolitestransferred from the picowell solution and absorbed into each cap canthen be analyzed. Preferably, the solution that is poured over thepicowell plate and over the bead becomes a hydrogel, and preferably thebead is made from a hydrogel.

In exclusionary embodiments, the present disclosure can exclude asystem, microtiter plate, microtiter plate with microwells, nanowells,or picowells, and related methods, where at least one well is capped,and where a liquid polymer solution is poured over the plate and overthe capped wells. Also, what can be excluded is the above where theliquid polymer has polymerized to form a solid polymer that adheres toeach cap. Also, what can be excluded is the method and resultingcompositions, where the solid polymer is torn away, removing with it theadhering caps.

FIG. 20 . Map of circular plasmid used for integrating IKZF1 gene intogenome of a cell. The plasmid is: IKZF1 mNEON-p2a-mScarlet-w3-2FB (9081base pairs). IKZF1 encodes the Ikarus protein.

FIG. 21 . Map of circular plasmid used for integrating IKZF3 gene intogenome of a cell. The plasmid is: IKZF3 mNeon-p2a-mScarlet-w3-2FB (9051bp). IKZF3 encodes the Aiolos protein.

FIG. 22 . Chemical monomers (compounds 1-6) and their DNA barcodes.

FIG. 23 . Chemical monomers (compounds 7-10) and their DNA barcodes.

FIG. 24 . Chemical monomers (compounds 11-16) and their DNA barcodes.

FIG. 25 . Chemical monomers (compounds 17-21) and their DNA barcodes.

FIG. 26 . Chemical monomers (compounds 22-16) and their DNA barcodes.

FIG. 27 Chemical monomers (compounds 27-30) and their DNA barcodes.

FIG. 28 . Sequencing a bead-bound DNA barcode. The figure disclosesintensity of fluorescent signal for each of five consecutive bases,where the five consecutive bases are part of a bead-bound DNA barcode.

FIG. 29 . Stepped picowell.

FIG. 30 . Time course of release of the fluorophore from the bead. Thisshows operation of the bead-bound release monitor, acquisition offluorescent data at t=0 seconds, t=1 seconds, t=11 seconds, and t=71seconds.

FIG. 31 . Emission data resulting after catalytic action of aspartylprotease on quencher-fluorophore substrate.

FIG. 32 . Drawings of cross-section of picowells, illustrating varioussteps.

FIG. 33 . Titration data showing how increase in UV dose results ingreater cleavage of fluorophore from the bead. In layperson's terms,this shows how a more powerful swing of the axe influences chopping thefluorophore from the bead (the power of the UV does is measured inJoules per centimeter squared). The notation “Exposure” refers only to aparameter when taking the photograph. It is just exposure time, whentaking the photograph (it does not refer to exposure time of the lightdoing the cleaving, or to the light doing the exciting).

FIG. 34 . TAMRA concentration versus luminous flux. What is shown isconcentration of free TAMRA, following release after exposure to UVlight at 365 nm.

FIG. 35 provides a hand-drawings of the quencher-fluorophore substrate,and of cleavage of this substrate by the enzyme, with consequentinhibition of enzyme. Also shown is the molecular structure ofbead-bound pepstatin-A, and bead-bound Fmoc-valine (negative control).

FIG. 36 . Steps in preparing beads for use in eventual capture of mRNAfrom lysed cells, with subsequent manufacture of cDNA library. Thisfigure also occurs in one of the Provisional applications (Compositionsand Method for Screening Compound Libraries on Single Cells), from whichpriority of the present application is claimed.

FIG. 37 . Tagging cells with DNA barcode, where tagging is by way of alipid that embeds in the cell membrane. This figure also occurs in oneof the Provisional applications (Compositions and Method for ScreeningCompound Libraries on Single Cells), from which priority of the instantapplication is claimed.

FIG. 38 is a schematic diagram of a computer device or system forperturbing a cell and capturing a response of the cell to theperturbation including at least one processor and a memory storing atleast one program for execution by the at least one processor accordingto an exemplary embodiment.

FIG. 39 is a side cross-sectional view of a dispenser with at least onecavity, and a sub-structure within the cavity according to an exemplaryembodiment.

FIG. 40 is a side cross-sectional view of a dispenser with at least onecavity, a tapered side wall, an open top end, and an open bottom endaccording to an exemplary embodiment.

FIG. 41 is a side cross-sectional view of a dispenser with at least onecavity, and a magnet disposed at the bottom of each cavity according toan exemplary embodiment.

FIG. 42 is a side cross-sectional view of a pipette with an optionalmesh insert according to an exemplary embodiment.

It is noted that the drawings are not necessarily to scale. The drawingsare intended to depict only typical aspects of the subject matterdisclosed herein, and therefore should not be considered as limiting thescope of the disclosure. Those skilled in the art will understand thatthe structures, systems, devices, and methods specifically describedherein and illustrated in the accompanying drawings are non-limitingexemplary embodiments and that the scope of the present invention isdefined solely by the claims.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the” include their corresponding pluralreferences unless the context clearly dictates otherwise. All referencescited herein are incorporated by reference to the same extent as if eachindividual patent, and published patent application, as well as figures,drawings, sequence listings, compact discs, and the like, wasspecifically and individually indicated to be incorporated by reference.

ABBREVIATIONS

Table 1 provides abbreviations and non-limiting definitions.

TABLE 1 Abbreviations and non-limiting definitions ACN AcetonitrileAMPSO 3-[(1,1-dimethyl-2-hydroxyethyl) amino]-2- hydroxypropanesulphonicacid. AMPSO is one of the “Good buffers” ((1966). Hydrogen Ion Buffersfor Biological Research. Biochemistry. 5:467-477). Aperture As usedherein, the term aperture is used herein to refer to a physicalsubstance that defines an opening and, more specifically, to the minimalamount of physical substance that is capable of defining an opening.Without implying any limitation, this minimal amount of physicalsubstance preferably takes the form of a ring-shaped section of a wall.Without limitation, the aperture can be considered to be a ring-shapedsection of a wall, where the thickness of the section is about 0.2 nm,about 0.5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about200 nm, about 500 nm, about 1 micrometer (um), about 2 um, about 5 um,and so on, where this thickness measurement is in the radial directionextending away from an axis, and where the axis is defined by theopening. 1-AP 1-Azidopyrene ATB Active tuberculosis Barcode The term“DNA barcode” can refer to a polynucleotide that identifies a chemicalcompound in its entirety while, in contrast, “DNA barcode module” canrefer to only one of the monomers that make up the chemical compound. Ashort definition of a “DNA barcode module” is that it identifies achemical library monomer. However, a “DNA barcode module” can be used toidentify the history of making that particular monomer. A longerdefinition of a “DNA barcode module” is as follows. Each of thefollowing chemical library monomers need to be identified by a different“DNA barcode module.” Even the first reaction and the second reactionhave the same reactants (A and B), a different DNA barcode module isused, because the products are different (the products being either C orD). Also, even though the first reaction and the third reaction resultin the same product (the product being “C”), a different DNA barcodemodule is used, because the reactants are different (the reactants beingeither A + B, or X + Y). Reaction Condition A + B → C Reaction conditionA, for example, with methane solvent A + B → D Reaction condition A, forexample, with methylene chloride solvent X + Y → C BiNAP BiNAP takes theform of two naphthalene groups attached to each other by way of acarbon-carbon bond between the 1-carbon of the first naphthalene and the1-carbon of the second naphthalene. Each naphthalene group also containsan attached PPh₂ group, where the PPh₂ group is attached to thenaphthalene’s 2-carbon. PPh₂ takes the form of a phosophate group, towhich is attached two phenyl groups. In BiNAP, the phosphate is situatedin between the naphthalene and the PPh₂. BTPBB Bis-Tris propane breakingbuffer BTPLB Bis-Tris propane ligation buffer BTPWB Bis-Tris propanewash buffer Cap A cap is an object that can serve as a plug, a stopper,a seal, and the like, for placing in stable contact with a microwell,nanowell, or picowell. The cap can be spherical, ovoid, cubical, cubicalwith rounded edges, pyramidal, pyramidal with rounded edges, and so on.Unless specified otherwise, the stated shape is the shape prior topartial insertion or prior to full insertion into the picowell.Preferably, when in use the cap is partially inserted into the picowellto form a seal. In some embodiments, the cap may be loosely set on topof the picowell without any partial insertion. Compound The term“compound” is used here, without implying any limitation, to refer to acompleted chemical that is synthesized by connecting a plurality ofchemical monomers to each other, by way of solid phase synthesis on abead. Generally, the term “compound” refers to the completed chemicalthat is to be tested for activity by way of an assay. The term“compound” is not intended to include any linkers that mediate bindingof the completed chemical to the bead, and is not intended to includeany protecting groups that are to be cleaved off, though it isunderstood that a “compound” that has a protecting group may havepharmaceutical activity. The term “compound” is NOT used to refer tobead-bound chemicals where not all of the chemical monomers have beenconnected. If the term “compound” is used in some other context herein,the skilled artisan will be able to determine if this description isrelevant or not. COMU 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (CAS no.1075198-30-9) Concatenated nucleic acid A DNA barcode that is“concatenated,” takes the form where all of the DNA barcode barcodemodules are part of the same polymer. When a bead contains a DNA barcodetaking the concatenated form, all of the information from all of theconstituent DNA barcode modules are present on the polymer that isattached to a single attachment site on the bead. Concatenated DNArefers to “end-to-end ligation” or “end-to-end joining” (Farzaneh (1988)Nucleic Acids Res. 16:11319- 11326, Boyer (1999) Virology. 263:307-312).In contrast, the word “catenated” refers to two circles of DNA that arelinked to each other as in a chain (Baird (1999) Proc. Nat’l. Acad. Sci.96:13685-13690). CuAAC Copper-catalyzed azide-alkyne cycloaddition CRBClick reaction buffer DAF Diazofluorene DBCO Dibenzocyclooctyne DBU1,8-diazabicyclo [5.4.0] undec-7-ene DCE 1,2-Dichloroethane DCMDichloromethane DESPS DNA encoded solid-phase synthesis DIC Diisopropylcarbodiimide DIEA N,N’-diisopropylethylamine DMA Dimethylacetamide DMAP4-Dimethylaminopyridine DMF Dimethylformamide DI Deionized DTTDithiothreitol EDC Ethyl-dimethylaminopropyl-carbodiimide ELISA Enzymelinked immunosorbent assay FMOC 9-Fluorenylmethoxycarbonyl FMOC-PCL-4-[4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]- OH2-methyoxy-5-nitrophenoxy] butanoic acid (CAS No. 162827-98-7)Functional In the context of a bead-bound DNA nucleic acid barcode, andin the context of manufacturing a bead-bound DNA barcode, the term“functional nucleic acid” refers to nucleic acids with an activebiochemical function (a function that takes advantage of hydrogen bonds,of hydrophobic interactions, of hydrophilic interactions, ofinteractions with enzymes, etc.). The function can be a spacer thatestablishes a distance between a hydrophobic bead and a primer bindingsite. The primer binding site preferably occurs in a hydrophilicenvironment for supporting activity of DNA polymerase. Also, thefunction can be a primer binding site, a hairpin bend, or the annealingsite for a “splint oligo.” This is in contrast to “informational nucleicacids,” which store information (which “encode”) information on theidentity of a corresponding chemical monomer. HDNA Headpiece DNA HTSHigh throughput screening INA 5-Iodonaphthalene-1-azide LC Liquidchromatography LTB Latent tuberculosis MDM2 Murine Double Minute 2 Mtt4-Methyltrityl NCL hits, NCL refers to a mixture of sera from latent NCLpool tuberculosis patients (this accounts for the letter “L”) and serafrom negative control, healthy human subjects (this accounts for theletter “NC”) NHS N-Hydroxysuccinimide. NHS chemistry can be used toattach tetrazine to free amino groups of, for example, antibodies (vanBuggenum, Gerlach, Mulder (2016) Scientific Reports. 6:22675. Nucleicacid The term “nucleic acid” can refer to a single nucleic acidmolecule, or to modified nucleic acids, such as a nucleic acid bearing afluorescent tag. Also, the term “nucleic acid” can be used to referindividual contiguous stretches of nucleotides within a longerpolynucleotide. Here, the term “nucleic acid” makes it more convenientto refer to these individual stretches within a longer polynucleotide,for example, as when the polynucleotide comprises a first nucleic acidthat is a primer-binding site, a second nucleic acid that is a DNAbarcode module, and a third nucleic acid that identifies the step numberin a multi-step pathway of synthesis. OP Oligo pair. Oligo pair canrefer to a reagent that takes the form of a slipped heteroduplex, forexample, an aqueous solution of a slipped heteroduplex. Orthogonal A DNAbarcode that is “orthogonal,” nucleic acid takes a form where each ofthe DNA barcode barcode modules occupies a different attachment site onthe bead. When a bead contains a DNA barcode taking the orthogonal form,the acquisition of all of the information of a compound’s DNA barcoderequires separately sequencing each of the attached DNA barcode modules.In other words, with an “orthogonal” nucleic acid barcode, each andevery one of the DNA barcode modules that makes up the DNA barcode isdispersed over different attachment sites on the same bead. OSu (OSu isN-Hydroxysuccinimide the same as NHS) OXYMA Ethyl2-cyano-2-(hydroxyamino)acetate Parallel The term “parallel” refers tothe situation where chemical monomers are covalently attached to a bead,one by one, to create a bead-bound compound, and where nucleic acidbarcode modules are also covalently attached to the same bead, one byone, to create a bead-bound nucleic acid barcode. The chemical reactionthat attaches each chemical monomer is not carried out at exactly thesame time as the reaction (chemical or enzymatic) that attaches eachnucleic acid barcode module. Instead, these two reactions are staggered,so that the parallel synthesis involves first attaching the chemicalmonomer, and then attaching the corresponding nucleic acid barcodemodule. Alternately, the staggered reaction can involve first attachingthe nucleic acid and then attaching the corresponding chemical monomer.What is corresponding in this situation, is that each nucleic acidbarcode module serves to identify the chemical monomer that is attachedin the same round of parallel synthesis. PCL Photocleavable linker PEGPolyethylene glycol Perturbation As used herein, the term “perturbation”is used broadly to encompass both small molecule chemical compounds,peptides, antibodies, macrocycles, or any library of molecules whoseeffect on cells, or in assays, is being interrogated. As used herein,the terms “perturbation beads” and “compound beads” are usedinterchangeably to denote beads that contain perturbations, as well asoligonucleotide barcodes that encode the synthesis history of theperturbations and/or the compound identity of the perturbations. As usedherein, the terms “compound-barcode” and “perturbation-barcode” are usedinterchangeably to denote the oligonucleotide tags that encode thesynthesis history and/or the identity of the compounds attached to theperturbation beads. PDMS Polydimethylsiloxane qPCR Quantitativepolymerase chain reaction Picowell Without implying any limitation onthe present disclosure, the term “picowell” can be used to refer to awell or cavity in a plate that contains an array of picowells, forexample, over 50,000 picowells, over 100,000 picowells, over 200,000picowells, over 500, 000 picowells, and so on. Typically, the volume ofa picowell (not including the volume of any beads that might be in thepicowell), is about 0.2 picoliters (pL), about 0.5 pL, about 1.0 pL,about 2.0 pL, about 5.0 pL, about 10 pL, about 20 pL, about 30 pL, about40 pL, about 50 pL, about 75 pL, about 100 pL, about 200 pL, about 300pL, about 400 pL, about 500 pL, about 600 pL, about 700 pL, about 800pL, about 1000 pL, about 10,000 pL, about 100,000 pL, about 1,000,000pL, or in a volume range defined by any of the above two values, forexample, about 0.5 to 2.0 pL. The volumes for any “nanowell” and“microwell” can be set as above (except with the term “pico” replaced bynano or micro). Unless specified otherwise, explicitly or by context,the present disclosure refers to picowells (rather than to nanowells ormicrowells). RAM Rink Amide RCA Rolling circle amplification RT Roomtemperature SPS Solid phase synthesis Slipped Slipped heteroduplexstructure takes the heteroduplex form of a first strand of ssDNA and astructure second strand of ssDNA, where a dozen nucleotides at the5’-end of the first strand of ssDNA are complementary to a dozennucleotides at the 5’-end of the second strand of ssDNA, and where thefirst strand of ssDNA is binds to the second strand of ssDNA by way of adozen complementary base pairings that involve the respective5’-termini. The number “dozen” is purely exemplary and is not limiting.Alternatively, the slipped heteroduplex structure could be maintained asa hybridized duplex, by way of complementary base pairing at the 3’-endof the first strand of ssDNA and the 3’-end of the second strand ofssDNA. The term “slipped heteroduplex structure” can alternatively becalled a “staggered heteroduplex structure.” The term “slipped” does notimply that the heteroduplex is slippery (can shift position) as might bethe case with a duplex formed when oligo[C] hybridizes to oligo[G], orwhen oligo[A] hybridizes to oligo[T]. TB Tuberculosis TBE Tris borateEDTA TBAI Tetrabutyl ammonium iodide. TBTATris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine TCEPTris(2-carboxyethyl)phosphine. Reducing agent that can cleave disulfidebonds. TCO Trans-cyclooctene TEAA Triethylammonium acetate TEV proteaseTobacco Etch Virus protease TFA Trifluoroacetic acid TID3-(trifluoromethyl)-3-(m-iodophenyl) diazirine TIPS Triisopropyl silaneTM Temperature of melting TMP 2,4,6-Trimethylpyridine QSY7 Xanthylium,9-[2-[[4-[[2,5-dioxo-1-pyrrolidinyl)oxy] carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6- bis(methylphenylamino)-, chloride (CAS No.304014-12-8) TAMRA 5(6)Carboxytetramethyl rhodamine

Reagents, kits, enzymes, buffers, living cells, instrumentation, and thelike, can be acquired. See, for example, Sigma-Aldrich, St. Louis, Mo.,Oakwood Chemical, Estill, S.C., Epicentre, Madison, Wis., Invitrogen,Carlsbad, Calif., ProMega, Madison, Wis., Life Technologies, Carlsbad,Calif., ThermoFisher Scientific, South San Francisco, Calif., NewEngland BioLabs, Ipswich, Mass., American Type Culture Collection(ATCC), Manassas, Va., Becton Dickinson, Franklin Lakes, N.J., Illumina,San Diego, Calif., 10× Genomics, Pleasanton, Calif.

Barcoded gel beads, non-barcoded gel beads, and microfluidic chips, areavailable from 1CellBio, Cambridge, Mass. Guidance and instrumentationfor flow cytometry is available (see, e.g., FACSCalibur®, BDBiosciences, San Jose, Calif., BD FACSAria II® User's Guide, part no.643245, Rev.A, December 2007, 344 pages).

A composition that is “labeled” is detectable, either directly orindirectly, by spectroscopic, photochemical, fluorometric, biochemical,immunochemical, isotopic, or chemical methods, as well as with methodsinvolving plasmonic nanoparticles. For example, useful labels include,³²P, ³³P, ³⁵S, ¹⁴C, ³H, ¹²⁵I, stable isotopes, epitope tags, fluorescentdyes, Raman tags, electron-dense reagents, substrates, or enzymes, e.g.,as used in enzyme-linked immunoassays, or fluorettes (Rozinov and Nolan(1998) Chem. Biol. 5:713-728).

  TABLE OF CONTENTS FOR DETAILED DESCRIPTION ( I ) Beads ( II ) One beadone compound (OBOC) ( III ) Coupling nucleic acids to beads ( IV ) DNAbarcodes ( V ) Coupling chemical compounds to beads ( VI ) Couplingchemical monomers to each other to make a compound ( VII ) Split andpool synthesis and parallel synthesis ( VIII ) Fabricating picowells (IX ) Deposit beads into picowells ( X ) Sequencing bead-bound nucleicacids in picowells ( XI ) Releasing bead-bound compounds from the bead (XII ) Biochemical assays for compounds ( XIII ) Cell-based assays forcompounds ( XIV ) Perturbation-response analysis on cells ( I ) BEADS

The methods and compositions of the present disclosure use beads, suchas monosized TentaGel® M NH₂ beads (10, 20, 30, etc., micrometers indiameter)-, standard TentaGel® amino resins (90, 130, etc. micrometersin diameter), TentaGel Macrobeads® (280-320 micrometers in diameter)(all of the above from Rapp Polymere, 72072 Tübingen, Germany). Thesehave a polystyrene core derivatized with polyethylene glycol (Paulick etal (2006) J. Comb. Chem. 8:417-426). TentaGel® resins are graftedcopolymers consisting of a low crosslinked polystyrene matrix on whichpolyethylene glycol (PEG) is grafted. Thus, the present disclosureprovides beads or resins that are modified to include one or both of aDNA barcode and a compound, where the unmodified beads take the form ofgrafted copolymers consisting of a low crosslinked polystyrene matrix onwhich polyethylene glycol (PEG) is grafted.

TentaGel® is characterized as, “PEG chains of molecular masses up to 20kilo Dalton have been immobilized on functionalized crosslinkedpolystyrenes. Graft copolymers with PEG chains of about 2000-3000 Daltonproved to be optimal in respect of kinetic rates, mobility, swelling andresin capacity.” (Rapp Polymere, Germany). Thus, the present disclosureprovides beads or resins that take the form of graft copolymers with PEGchains of about 2000-3000 Daltons. Regarding swelling, Comellas et alprovides guidance for measuring the ability of a bead to swell, forexample, when soaked in DCM, DMF, methyl alcohol, water, or a bufferused in enzyme assays (Comellas et al (2009) PLoS ONE. 4:e6222 (12pages)). The unit of swelling is milliliters per gram of bead.

In an alternate bead embodiment, the present disclosure uses a resinwith a PEG spacer is attached to the polystyrene backbone via an alkyllinkage, and where the resin is microspherical and monosized (TentaGel®M resin).

In yet an alternate bead embodiment, the present disclosure uses a resinwith a PEG spacer attached to the polystyrene backbone via an alkyllinkage, where the resin type exists in two bifunctional species: First,surface modified resins: the reactive sites on the outer surface of thebeads are protected orthogonally to the reactive sites in the internalvolume of the beads, and second, hybrid resins: cleavable andnoncleavable ligands are present in this support—developed forsequential cleavage (TentaGel® B resin).

Moreover, in another embodiment, the present disclosure uses a resinwhere a PEG spacer is attached to the polystyrene backbone via an alkyllinkage, and where the macrobead resin shows very large particlediameters and high capacities (TentaGel® MB resin). Also, the presentdisclosure uses a resin where the PEG spacer is attached to thepolystyrene backbone via a benzyl ether linkage. This resin can be usedfor immunization procedures or for synthesizing PEG modified derivatives(PEG Attached PEG-modified compounds) (TentaGEl® PAP resin).

Moreover, the beads can be, HypoGel® 200 resins. These resins arecomposites of oligoethylene glycol (MW 200) grafted onto a lowcross-linked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs,Switzerland).

In some embodiments amino functionalized polystyrene beads, without PEGlinkers, may be used, for instance, monosized polystyrene M NH₂microbeads (5, 10, 20 etc., micrometers in diameter, also from RappPolymere, 72072 Tübingen, Germany).

In some embodiments, compounds may be encapsulated within pores orchambers or tunnels within the beads, without covalent attachment to thebeads. Compounds may be diffused into or forced within such pores of thebeads by various means. In some embodiments the compounds may be loadedwithin the beads by diffusion. In some embodiments, high temperature maybe used to swell the beads and load compounds within the beads. In someembodiments, high pressure may be used to force compounds into thebeads. In some embodiments, solvents that swell the beads may be used toload compound within the beads. In some embodiments, vacuum or lowpressure may be used to partition compounds into beads. In someembodiments mild, or vigorous physical agitation may be used to loadcompounds into beads.

In such embodiments where the compounds are loaded onto beads withoutcovalent attachment, compounds may be unloaded from the bead by way ofdiffusion. In some embodiments, in a non-limiting fashion, temperature,pressure, solvents, pH, salts, buffer or detergent or combinations ofsuch conditions may be used to unload compounds out of such beads. Insome embodiments the physical integrity of the beads, for instance byuncrosslinking polymerized beads, may be used to release compoundscontained within such beads.

In exclusionary embodiments, the present disclosure can exclude anybead, and bead-compound complex, or any method, that involves one of theabove beads.

Beads of the present disclosure also include the following. Merrifieldresin (chloromethylpolystyrene), PAM resin (4-hydroxymethylphenylacetamido methyl polystyrene), MBHA resin (4-methylbenzhydrylamine),Brominated Wang resin (alpha-bromopriopiophenone), 4-Nitrobenzophenoneoxime (Kaiser) resin, Wang resin (4hydroxymethyl phenoxymethylpolystyrene, PHB resin (p-hydroxybenzyl alcohol, HMPA resin(4-hydroxymethyl phenoxyacetic acid), HMPB resin(4-hydroxymethyl-3-methoxy phenoxyl butanoic acid), 2-Chlorotritylresin, 4-Carboxytrityl resin, Rink acid resin (4-[(2,4-dimethoxypehenyl)hydroxymethyl) phenoxymethyl), Rink amide (RAM) resin “Knorr” resin(4-((2,4-dimethylphenyl) (Fmox-amino)methyl) phenoxyalkyl), PAL resin(5-[4-(Fmoc-amino) methyl-3,5-dimethoxyphenoxy] valeramidomethylpolystyrene), Sieber amide resin (9-Fmox-amino-xanthan-3-yl-oxymethyl),HMBA resin (hydroxymethyl benzoic acid), 4-Sulfamoylbenzoyl resin“Kenner's safety catch” resin (N-(4-sulfamoylbenzoyl)aminomethyl-polystyrene), FMP-resin(4-(4-formyl-3-methoxyphenoxy)-ethyl) (see, ChemFiles Resins forSolid-Phase Peptide Synthesis Vol. 3 (32 pages) (Fluka Chemie GmbH,CH-9471 Buchs, Switzerland).

Beads of the present disclosure further include the above beads used aspassive encapsulants of compounds (passively hold compounds withoutcovalent linkage to the compound), and further comprising the following:unfunctionalized polystyrene beads, silica beads, alumina beads, porousglass beads, polyacrylamide beads, titanium oxide beads, alginate beads,ceramic beads, PMMA (polymethylmethacrylate) beads, melamine beads,zeolite beds, polylactide beads, deblock-copolymer micelles, dextranbeads, and others. Many of the beads listed in this paragraph may bepurchased from vendors such as Microspheres-Nanospheres, Cold Spring,N.Y. 10516, USA.

In addition to beads, vesicles or droplets may also be used as vehiclesfor delivering compounds for some embodiments of the present disclosure.Lipids, deblock-copolymers, tri-block copolymers or other membraneforming materials may be used to form an internal volume into whichcompounds may be loaded. Compounds may be released from theseencapsulated volumes by addition of detergent, mechanical agitation,temperature, salt, pH or other means. Water-in-oil droplet emulsions oroil-in-water droplet emulsions are yet other means to passivelyencapsulate compounds that may be delivered to assay volumes.

In all embodiments where passive encapsulation is used to delivercompounds, DNA tags may also be loaded passively, or alternatively, theDNA tags may be covalently attached to the beads, vesicles or droplets.

In exclusionary embodiments, the present disclosure can exclude anybeads or resins that are made of any one the above chemicals, or thatare made of derivatives of one any one of the above chemicals.

In embodiments, the beads can be spheroid and have a diameter of about0.1-1 micrometers, about 1-5 micrometers, about 1-10, about 5-10, about5-20, about 5-30, about 10-20, about 10-30, about 10-40, about 10-50,about 20-30, about 20-40, about 20-50, about 20-60, about 50-100, about50-200, about 50-300, about 50-400, about 100-200, about 100-400, about100-600, about 100-800, about 200-400, about 200-600, about 200-800micrometers, and so on.

Non-spheroid beads that are definable in terms of the above values andranges are also provided. For example, one of the axes, or one of theprimary dimensions (for example, a side) or one of the secondarydimensions (for example, a diagonal) may comprise values in the aboveranges. In exclusionary embodiments, the present disclosure can excludeany reagent, composition, system, or method, that encompasses spheroidbeads (or non-spheroid beads) falling into one or more of the abovevalues or ranges.

Chains of beads. In one embodiment, what is provided is a plurality ofbead dimers, where the bead-dimer takes the form of two beads that areattached to each other, and where one bead contains a plurality ofattached nucleic acid barcodes (either orthogonal nucleic acid modules,or concatenated nucleic acid modules), and the other bead contains aplurality of attached compounds, where all of the compounds aresubstantially related to each other (or where all of the compounds aresubstantially identical in chemical structure to each other). The beaddimer may be synthesized by preparing the first bead that has theattached compounds, separately preparing the second bead that hasattached nucleic acid barcodes, and then linking the two beads together.In one aspect, the beads are attached to each other by a reversiblelinker, and in another aspect, the beads are attached to each other by anon-reversible linker.

Bead permeability. In embodiments, the present disclosure provides beadswith various ranges or degrees of permeability. Permeability can bemeasured as the percentage of the volume of the bead that is accessibleby a solvent, where the unit of measurement is percentage of the bead'ssurface that takes the form or pores, or where the unit of measurementis percentage of the bead's interior that takes the form of channels,networks, or chambers that are in fluid communication with the surface(and exterior medium) of the bead. The present disclosure can encompassporous beads or, alternatively, can exclude porous beads.

U.S. Pat. No. 9,062,304 of Rothberg discloses a bead with an exteriorand with interior regions. What is shown is “internal surfaces (poresurfaces),” and that “suitable pores will . . . exclude largermolecules,” and the option of “exploiting differential functionalizationof interior and exterior surfaces,” and various pore diameters, andpolymers such as poly(styrene sulfonic acid) and polystyrene. FIG. 1 ofRothberg provides pictures of surface of bead and pores of bead. U.S.Pat. No. 9,745,438 of Bedre provides transmission electron microscopeimage of porous bead. U.S. Pat. No. 5,888,930 of Smith provides scanningelectron micrograph of cross-section of porous bead. What is shown isspherical bead with small pores on surface and large pores inside, wherebead is made from, e.g., polystyrene, polyacrylonitrile, polycarbonate,cellulose, or polyurethane. U.S. Pat. No. 5,047,437 of Cooke disclosesspherical poly(acrylonitrile) copolymer pore morphology with skinlesssurface (FIG. 1) and bead that has exterior skin on surface (FIG. 5).U.S. Pat. No. 4,090,022 of Tsao discloses porous openings and internalvoid spaces, of cellulose beads.

Each of the above-identified patents, including all of the figures, isincorporated herein in its entirety, as though each was individuallyincorporated by reference in its entirety.

Without implying any limitation, exterior surface of a bead ormicroparticle can be determined by tightly wrapping the entire bead ormicroparticle with an elastic film. The bead or microparticle can bewrapped by way of a thought-experiment, or the wrapped bead can bedepicted by a drawing or photograph, or the bead can be wrapped inreality. Without implying any limitation, the exterior surface of thebead is that part of the bead that physically contacts the wrapping.

For example, the present disclosure provides a bead with poresaccounting for at least 1%, at least 2%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, of the surfacearea. Also, the present disclosure provides a bead where the volume ofthe internal channels or networks accounts for at least 1%, at least 2%,at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, ofthe total volume of the bead, and where the internal channels ornetworks are in fluid communication with the outside surface (andexterior medium) of the bead.

Moreover, the present disclosure provides a bead with pores accountingfor less than 1%, less than 2%, less than 5%, less than 10%, less than15%, less than 20%, less than 30%, less than 40%, of the surface area.Also, the present disclosure provides a bead where the volume of theinternal channels or networks accounts for less than 1%, less than 2%,less than 5%, less than 10%, less than 15%, less than 20%, less than30%, less than 40%, less than 50%, less than 60%, less than 70%, lessthan 80%, of the total volume of the bead, and where the internalchannels or networks are in fluid communication with the outside surface(and exterior medium) of the bead.

Iron-core beads. The present disclosure encompasses iron-core beads ormagnetic beads. These beads can be manipulated with magnets to move themfrom one reaction vessel to another reaction vessel, or from onecontainer to another container. Manipulations by robotics can beenhanced by using these beads. Methods of manufacture and use ofmagnetic beads are available (Szymonifka and Chapman (1994) TetrahedronLetters. 36:1597-1600, Liu, Qian, Xiao (2011) ACS Comb. Sci. 13:537-546,Alam, Maeda, Sasaki (2000) Bioorg. Med. Chem. 8:465-473).

In exclusionary embodiments, the present disclosure can exclude anybead, or any population of beads, where the bead or population meets oneof the above values or ranges.

Compound Loading into Beads

In many experiments it is advantageous to load pre-synthesized compoundsinto beads, where the beads may be used as vehicles for delivering thecompounds to an assay. Many of the standard techniques used for drugdelivery to biological specimens may be adapted to deliver compounds toassays (see, Wilczewska et al (2012) Nanoparticles as drug deliverysystems. Pharmacological Reports. 64:1020-1037, Kohane D S (2007)Microparticles and nanoparticles for drug delivery. Biotechnol. Bioeng.96: 203-209, Singh et al (2010) Microencapsulation: A promisingtechnique for controlled drug delivery. Res Pharm Sci. 5: 65-77).

In such embodiments where pre-synthesized compounds are loaded intobeads, the compounds may be held in traditional 96, 385 or 1536 wellmicrotiter plates. To these plates, beads may be added, into which thecompounds get loaded by diffusion or by other active loading methods. Inpreferred embodiments, the beads chosen for impregnation have pore sizesor percolation geometries that prevent immediate emptying of thecompounds when removed from the mother solution. The diffusion out ofthe beads may be enhanced by heat, pressure, additives or otherstimulants, if needed. In some embodiments, the compound-laden beads maybe capped in a manner that prevents leakage of the internal contentsuntil triggered by an external impulse. One method for capping theexteriors of porous beads involves adding lipids or amphiphilicmolecules to the bead-compound solution, such that the cavities exposedto the surface of the beads get sealed by a bilayer formed by theamphiphilic molecules. In some embodiments preformed vesicles may bemixed with the drug laden beads, such that upon agitating, the vesiclesrupture and the membranes reform over the surface of the drug-ladenbeads, thereby sealing them. Methods to perform such bead sealing aredescribed (see, Tanuj Sapra et al (2012) Nature Scientific Reportsvolume 2, Article No.: 848). Further experimental protocols to sealsilica beads are available in the report release by Sandia Laboratoriesby Ryan Davis et al, Nanoporous Microbead Supported Bilayers: Stability,Physical Characterization, and Incorporation of Functional TransmembraneProteins, SAND2007-1560, and the method bSUM, described by Hui Zheng etal (bSUM: A bead-supported unilamellar membrane system facilitatingunidirectional insertion of membrane proteins into giant vesicles) in J.Gen. Physiol. (2016) 147: 77-93.

In some embodiments utilizing pre-synthesized compounds, beads aregenerated from the compounds by addition of appropriate reagents, forinstance by adding lipids or di-block copolymers followed by agitation,whereby vesicles are formed containing the compounds in their interioror within the bilayer membrane. In some embodiments, the compounds maybe pushed through a microfluidic T junction to create aqueous phasedroplets in an oil phase, where the compounds are contained within theaqueous phase or at the interface between the aqueous phase and the oilphase. In some embodiments, the droplets formed may further bepolymerized, creating hydrogels, that are more rugged and stable tohandling than unpolymerized aqueous phase droplets. Droplet-basedencapsulation and assays are disclosed by, Oliver et al (2013) DropletBased Microfluidics, SLAS Discovery Volume: 19 issue: 4, page(s):483-496. Sol-gel encapsulation process may also be employed toencapsulate compounds within beads. Formation of sol-gel beads isdescribed in, Sol-gel Encapsulation of Biomolecules and Cells forMedicinal Applications, Xiaolin Wang et al (2015) Current Topics inMedicinal Chemistry. 15: 223.

One Bead One Compound (OBOC)

Methods used to manufacture combinatorial libraries involve three steps,

(1) Preparing the library, (2) Screening the compounds in the library,and (3) Determining the structure of the compounds, for example, of allof the compounds or only of the compounds that provided an interestingresult with screening (see, Lam et al (1997) The One-Bead-One-CompoundCombinatorial Library Method. Chem. Rev. 97:411-448). An advantage ofsynthesizing compounds by way of a bead-bound synthesis, is that thecompound can be made rapidly by the “split-and-pool” method.

OBOC combined with an encoding strategy. Another feature of OBOC is thateach bead can include, not only a compound but also an encodingstrategy. Where bead-bound nucleic acids are used for encoding acompound that is bound to the same bead, the term “encoding” does NOTrefer to the genetic code. Instead, the term “encoding” means that theuser possesses a legend, key, or code, that correlates each of manythousands of short nucleic acid sequences with a single bead-boundcompound.

A dramatic variation of using a bead that bears bead-bound compounds andbead-bound nucleic acids, where the nucleic acids encode the associatedcompounds, is as follows. The dramatic variation is to manufacture alibrary of conjugates, where each member of the library takes the formof a conjugate of a small molecule plus a DNA moiety, where the DNAmoiety encodes the small molecule). This conjugate is soluble and is notbead-bound. After screening with a cell or with a purified protein, theconjugate remains bound to the cell or purified protein, therebyenabling isolation of the conjugate and eventual identifying thecompound by sequencing the conjugated nucleic acid (see, Satz et al(2015) Bioconjugate Chemistry. 26:1623-1632).

Here, as in most of this patent document, the term “encode” does notrefer to the genetic code, but instead it refers to the fact that theresearcher uses a specific nucleic acid sequence to indicate a specific,known structure of a compound that is attached to it.

As an alternative to using an encoding strategy, such as the use of aDNA barcode, a bead that screens positive (thereby indicating a compoundthat screens positive) can be subjected to Edman degradation or to massspectrometry to identify the bead-bound compound (see, Shih et al (2017)Mol. Cancer Ther. 16:1212-1223). If the bead-bound compounds arepeptides, then MALDI mass spectrometry can be used for directdetermination of the sequence of a positively-screening peptidecompound. Direct sequencing is possible, because simultaneous cleavageand ionization occur under laser irradiation (Song, Lam (2003) J. Am.Chem. Soc. 125:6180-6188).

One fine point in performing split-and-pool synthesis of a combinatoriallibrary, is that the compound can be manufactured so that all of thecompounds share a common motif. This strategy has been described as the,“generation of a library of motifs rather than a library of compounds”(see, Sepetov et al (1995) Proc. Natl. Acad. Sci. 92:5426-5430, Lam etal, supra, at 418).

To provide a typical example of a large bead, the bead can be 0.1 mm indiameter and it can hold about 10¹³ copies of the same compound (Lam etal, supra). Following preparation of a library of bead-bound compounds,each bead can be used in individual assays, where the assays measurebiochemical activity or, alternatively, a binding activity. Assays canbe “on-bead” assays or, alternatively, the compound can be severed fromthe bead and used in solution-phase assays (Lam et al, supra).

Parameters of any type of bead include its tendency to swell in a givenassay medium, whether the bead's polymer is hydrophobic or hydrophilic,the identity of the attachments sites on the bead for attaching eachcompound, the issue of whether a spacer such as polyethylene glycol(PEG) is used to provide some separation of each compound from thebead's surface, and the internal volume of the bead.

Regarding the need to attach compounds to the bead, but at a distancefar away from the bead's hydrophobic surface, Lam et al, supra,discloses that polyoxyethylene-grafted styrene (TentaGel®) has theadvantage that the functionalizable group is at the end of apolyoxyethylene chain, and thus far away from the hydrophobicpolystyrene. Beads that possess a water-soluble linker include TentaGeland polydimethylacrylamide bead (PepSyn® gel, Cambridge ResearchBiochemicals, Northwitch, UK).

The parameter of internal volume can provide an advantage, where thereis a need to prevent interactions between the bead-bound DNA barcode andthe target of the bead-bound compound. To exploit this advantage, thebead can be manufactured so that the DNA barcode is situated in theinside of the bead while, in contrast, the compound that is beingscreened is attached to the bead's surface (Lam et al, supra, at pages438-439). This advantage of internal volume may be irrelevant, where thebead-bound compound is attached by a cleavable linker, and where assaysof the compound are conducted only on compounds that are cleaved andreleased.

Appell et al, provide a non-limiting example of spit-and-pool method forsynthesizing a chemical library followed by screening to detect activecompounds (Appell et al (1996) J. Biomolecular Screening. 1:27-31).Library beads are placed, one into each well, in an array of wells on afirst microwell plate, nanowell plate, or picowell plate. Beads areexposed to light, in order to cleave about 50% of the bead-boundcompounds, releasing them into solution in the well. Released compoundis then transferred to a second microwell plate, and subjected to assaysfor detecting wells that contain active compounds, thereby identifyingwhich beads in the first plate contain bead-bound compounds that areactive. Then, “[o]nce an active [compound] is identified from a singlebead, the bead is recovered and decoded, thus yielding the synthetichistory and . . . structure of the active compound” (Appell et al,supra).

For cell-based screening assays that screen for bead-bound compounds,Shih et al provide a novel type of bead (Shih et al (2017) Mol. CancerTher. 16:1212-1223). This novel type of bead contains a bead-boundcompound that is a member of a library of “synthetic death ligandsagainst ovarian cancer.” The bead is also decorated with biotin, wheretwo more chemicals are added that create a sandwich, and where thesandwich maintains adhesion of the cell to the bead. The sandwichincludes a streptavidin plus biotin-LXY30 complex. This sandwichconnects the bead to LXY30's receptor, which happens to be a well-knownprotein on the cell surface, namely, an integrin. The method of Shih etal, supra, resulted in the discovery of a new molecule (“LLS2”) that cankill cancer cells. The above method uses bead-bound compounds, where thecompounds bind to cells (even though the compound is still bead-bound).Cho et al created a similar one-bead-one-compound library, where thecompound being screened was sufficient to bind to cells (without anyneed for the above-described sandwich) (Cho et al (2013) ACSCombinatorial Science. 15:393-400). The goal of the Cho et al, reportwas to discover RGD-containing peptides that bind to integrin that isexpressed by cancer cells. The above-disclosed reagents and methods areuseful for the present disclosure.

Coupling Nucleic Acids to Beads (Orthogonal Style, Concatenated Style)

One way to get oriented to the topic of concatenated barcodes andorthogonal barcodes, is to note advantages that one has over the other.An advantage of orthogonal barcoding over concatenated barcoding, is asfollows. With attachment of each monomer of a growing chemical compound,what is attached in parallel is a DNA barcode module. With concatenatedbarcoding, if attachment of any given module is imperfect (meaning, thatnot all of the attachments sites was successfully coupled with a neededmodule), then the sequence of the completed barcode will not be correct.The statement “not be correct” means that imperfect coupling means thatchunks may be missing from wad was assumed to be the completed, correctDNA barcode. Here, the completed barcode sequence will contain amistake, due to failure of attachment of all of the modules. Incontrast, with orthogonal barcoding each individual module getscovalently bound to its own unique attachment site on the bead. Andwhere once a module gets attached to a given site on the bead, nofurther modules will be connected to the module that is alreadyattached.

The present disclosure provides reagents and methods for reducing damageto bead-bound DNA barcodes, and for reducing damage to partiallysynthesized bead-bound DNA barcodes. Each DNA barcode module, prior toattaching to a growing bead-bound DNA barcode, can take the form ofdouble stranded DNA (dsDNA), where this dsDNA is treated with a DNAcross-linker such as mitomycin-C. After completion of the synthesis ofthe DNA barcode in its dsDNA form, this dsDNA is converted to ssDNA.Conversion of dsDNA to ssDNA can be effected where one of the DNAstrands has a uracil (U) residue, and where cleavage of the DNA at theposition of the uracil residue is catalyzed by uracil-N-glycosidase(see, FIG. 5 of Ser. No. 62/562,905, filed Sep. 25, 2017. Ser. No.62/562,905 is incorporated herein by reference in its entirety). Theabove refers to damage that is inflicted on the growing DNA barcode byreagents used to make the bead-bound chemical compound.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is bysynthesizing the DNA barcode in a double stranded DNA form, where eachof the DNA barcode modules that are being attached to each other takesthe form of dsDNA, and where each of the two strands is stabilized byway of a DNA headpiece. For eventual sequencing of the completed DNAbarcode, one of the strands is cleaved off from the DNA headpiece andremoved. The above refers to damage that is inflicted on the growing DNAbarcode by reagents used to make the bead-bound chemical compound (wherethis chemical compound is a member of the chemical library).

Yet another method for reducing damage to bead-bound DNA barcodes, is tosynthesize the DNA barcode in a way that self-assembles to form ahairpin, and where this DNA barcode self-assembles to that the firstprong of the hairpin anneals to the second prong of the hairpin.

Where the DNA barcode being synthesized takes the form of doublestranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denaturethe DNA barcode. The above methods and reagents can preventdenaturation.

As stated above, the term “DNA barcode” can refer to a polynucleotidethat identifies a chemical compound in its entirety while, in contrast,“DNA barcode module” can refer to only one of the monomers that make upthe chemical compound.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is to use doublestranded DNA (dsDNA) and to seal the ends of this dsDNA by way of7-aza-dATP and dGTP.

In alternate embodiments, the method can use an intermediate between“concatenated DNA barcoding” and “orthogonal DNA barcoding,” where thisintermediate involves blocks of DNA barcodes, that is, where each blockcontains two DNA modules, or contains three DNA modules, or containsfour DNA modules, or contains five DNA modules, and the like (but doesnot contain all of the DNA modules that identify the full-lengthcompound).

FIG. 1 discloses an exemplary and non-limiting diagram of theCONCATENATED structured bead. The bead contains a plurality of DNAbarcodes (each made of DNA barcode modules) and a plurality of compounds(each made of chemical library monomers). For ease in speaking, the term“DNA barcode” may be used to refer to the polymer that includes all ofthe nucleic acids that are a “DNA barcode module,” as well as all of thenucleic acids that provide some function. The function can be anannealing site for a sequencing primer, or the function can be used toidentify a step in chemical synthesis of the bead-bound compound. FIG. 1also shows bead-bound compounds, where each compound is made of severalchemical library members, and where each chemical library member isrepresented by a square, circle, or triangle. FIG. 1 shows that each DNAbarcode module is numbered, consecutively, from 1 to 8, where thesenumbers correspond to the respective eight shapes (squares, circles,triangles). For clarity, nucleic acids that serve a function (and do notrepresent or “encode” any particular chemical unit) are not shown in thefigure.

FIG. 2 discloses an exemplary and non-limiting embodiment of theORTHOGONAL structured bead. The bead contains a plurality of DNAbarcodes (each made of DNA barcode modules), but each DNA barcode moduleis attached to a separate linking site on the bead. The entire DNAbarcode consists of eight DNA barcode modules, which in the figure arenumbered 1-8. When the information from a particular DNA barcode isread, and then used to identify the chemical compound that is bound tothe same bead, one must perform DNA sequencing on each of the separatelyattached DNA barcode modules. In FIG. 2 , the bead also contains aplurality of attached chemical compounds, each with eight units, asshown by the eight shapes (circles, squares, triangles).

In FIG. 2 , for clarity, functional nucleic acids that are attached toeach DNA barcode module is not shown. Of course, each of the DNA barcodemodules needs to have a nucleic acid that identifies the position of thechemical library monomer in the completed, full-length compound. For theexample shown in FIG. 2 , the position needs to be first, second, third,fourth, fifth, sixth, seventh, or eighth.

In one embodiment, the chemical monomer is first attached and then,after that, the corresponding DNA barcode module is attached. In analternative embodiment, the DNA barcode module is first attached, andthen the corresponding chemical monomer is attached. Also, a procedureof organic synthesis can be followed that sometimes uses the “oneembodiment” and sometimes uses the “alternative embodiment.” In yetanother alternative embodiment, the present method provides block-wiseaddition of a block of several chemical monomers which is attached tothe bead, in parallel with attachment of a block of several DNA barcodemodules.

In exclusionary embodiments, what can be excluded is reagents,compositions, and methods that used block-wise addition of chemicalmonomers, of DNA barcode modules, or of both chemical monomers and DNAbarcode modules, to a bead.

This concerns nucleic acids that may be present in the bead-boundpolynucleotide, including nucleic acids that “encode” or serve toidentify monomers of a bead-bound compound. In exclusionary embodiments,the present disclosure can exclude a nucleic acid that encodes a“step-specific DNA sequencing primer site.” In this situation, for eachchemical monomer that is present in a compound, there is a correspondingDNA barcode module, where each DNA barcode module is flanked by at leastone corresponding primer-binding site, that is, “a step-specific DNAsequencing primer site.” Also, what can be excluded is a nucleic acidthat encodes or designates a particular step in the chemical synthesisof a compound, such as step 1, step 2, step 3, or step 4.

Moreover, the present disclosure can include a nucleic acid thatfunctions as a spacer. For example, as spacer can create a distance,along a polynucleotide chain, between a first site that is a sequencingprimer annealing site and a second site that identifies a chemicalmonomer. Also, the present disclosure can use a nucleic acid thatreiterates or confirms the information provided by another nucleic acid.Also, the present disclosure can use a nucleic acid that encodes a PCRprimer binding site. A PCR primer binding site can be distinguished froma sequencing primer, because a polynucleotide with a PCR primer bindingsite has two PCR primer binding sites, and because both of these sitesare designed to have the same melting point (melting point when the PCRprimer is annealed to PCR primer binding site).

In exclusionary embodiments, the present disclosure can exclude anucleic acid that functions as a spacer, or solely as a spacer. Also,the present disclosure can exclude a nucleic acid that reiterates orconfirms the info provided by another nucleic acid. Moreover, thepresent disclosure can exclude a nucleic acid that serves as a PCRprimer binding site, and can exclude a nucleic acid that serves as abinding site for a primer that is not a PCR primer.

Additionally, the present disclosure can exclude a nucleic acid thatidentifies the date that a chemical library was made, or that identifiesa step in chemical synthesis of a particular compound, or that serves asa primer annealing sequence.

Dedication of sequencing primers to a particular DNA barcode module. Thepresent disclosure provides a DNA barcode that contains DNA barcodemodules and one or more sequencing primer annealing sites. Each DNAbarcode module may have its own, dedicated, sequencing primer bindingsite. Alternatively, one particular sequencing primer binding site maybe used for sequencing two, three, four, five, 6, 7, 8, 9, 10, or moreconsecutive DNA barcode modules, as may exist on the bead-bound DNAbarcode.

The following describes the situation where each DNA barcode module hasits own dedicated sequencing primer binding site. The present disclosureprovides a bead-bound concatenated barcode comprising a primer bindingsite capable of binding a DNA sequencing primer, wherein said primerbinding site is capable of directing sequencing of one or more of the1^(st) DNA barcode module, the 2^(nd) DNA barcode module, the 3^(rd) DNAbarcode module, the 4^(th) DNA barcode module, the 5^(th) DNA barcodemodule, and the 6^(th) DNA barcode module, and wherein the primerbinding site is situated 3-prime to the 1^(st) DNA barcode module withno other DNA barcode module in between the 1^(st) DNA barcode module andthe primer binding site, 3-prime to the 2^(nd) DNA barcode module withno other DNA barcode module in between, 3-prime to the 3^(rd) DNAbarcode module with no other DNA barcode module in between, 3-prime tothe 4^(th) DNA barcode module with no other DNA barcode module inbetween, 3-prime to the 5^(th) DNA barcode module with no other DNAbarcode module in between, or 3-prime to the 6^(th) DNA barcode modulewith no other DNA barcode module in between.

Encoding sequences and sequences complementary to encoding sequences.The present disclosure can encompass any one, any combination of, or allof the encoding sequences disclosed above, or elsewhere, in thisdocument. In exclusionary embodiments, what can be excluded are any one,any combination of, or all of the encoding sequences disclosed above, orelsewhere, in this document. What can also be included or can beexcluded are double stranded nucleic acids that encode any one, anycombination of, or all of the encoding sequences described above, orelsewhere, in this document.

Orthogonal-Style DNA Barcode (Each DNA Barcode Module Attached toSeparate Location on Bead)

Synthesis of orthogonal-style bead. With orthogonal synthesis, each DNAmodule gets covalently attached to a separate site on the bead, andwhere the result is that the entire DNA barcode is contributed by aplurality of DNA modules. Where the DNA barcode has the orthogonalstructure, none of the DNA barcode modules are attached to eachother—instead each and every one of the DNA barcode molecules has itsown bead-attachment site that is dedicated to that particular DNAbarcode module.

Nucleic acid identifying the synthesis step number for each DNA barcodemodule. In embodiments, the orthogonal DNA barcode includes a shortnucleic acid that identifies the first step of compound synthesis. Forthis embodiment, with the parallel attachment of the first chemicalmonomer and the first DNA barcode module, the first DNA barcode moduleactually takes the form of this complex of two nucleic acids: [SHORTNUCLEIC ACID THAT MEANS “STEP ONE” ] connected to [FIRST DNA BARCODEMODULE]. All of the nucleotides of this complex are in-frame with eachother and can be read in a sequencing assay, but the first short nucleicacid may optionally be attached to the first DNA barcode module by wayof a spacer nucleic acid.

The following continues the above description of the orthogonal DNAbarcode. The orthogonal DNA barcode includes a short nucleic acid thatidentifies the second step of compound synthesis. For this embodiment,with the parallel attachment of the second chemical monomer and thesecond DNA barcode module, the second DNA barcode module actually takesthe form of this complex of two nucleic acids: [SHORT NUCLEIC ACID THATMEANS “STEP TWO” ] connected to [SECOND DNA BARCODE MODULE]. All of thenucleotides of this complex are in-frame with each other and can be readin a sequencing assay, but the second short nucleic acid may optionallybe attached to the second DNA barcode module by way of a spacer nucleicacid.

The above-described method is repeated for the third, fourth, fifth,sixth, seventh, eighth, ninth, tenth, and up to the last of the DNAbarcode modules and up to the last of the chemical monomers, for anygiven bead. The above-method can be followed when using split-and-poolsynthesis, for creating DNA barcodes and chemical compounds that arebead-bound.

The orthogonal structure provides the following advantage over theconcatenated structure. With concatenated synthesis (all DNA barcodemodules attached to each other in one, continuous polymer) it is thecase that failure to achieve synthesis any of the intermediates couplingsteps can ruin the meaning of the concatenated DNA barcode that iseventually completed. In contrast, with orthogonal synthesis (each andevery one of the DNA barcode modules attached to a dedicated site on thebead), failure to attach any of the DNA barcode modules will only resultin an empty attachment site on the bead, and will not ruin the meaningof any of the other attached DNA barcode modules. In a preferredembodiment, each attached DNA barcode module includes an attached,second nucleic acid, where this second nucleic acid identifies the step(the step during the parallel synthesis of DNA barcode and chemicalcompound).

For orthogonal synthesis, it is acceptable for all of the attachmentsites on the bead to be used up (sites for attaching the growingchemical library member). However, for orthogonal synthesis, thechemical reaction needs to be designed so that the entire population ofattachment sites on the bead is only partly used up, with attachment ofthe first of many DNA barcode modules. The following provides optionallimits for using up sites during chemical synthesis of an orthogonalbarcode. For the non-modified bead, the total number of sites availablefor attaching a DNA barcode module is 100%.

Extent of using up attachment sites on a given bead, with synthesis ofan orthogonal-configured bead (regarding the 1^(st) DNA barcode). Thefollowing concerns attaching the first DNA barcode module. Inembodiments, with attachment of the first DNA barcode module, about 5%,about 10%, about 20%, about 30%, about 40%, or about 50% of the DNAbarcode attachments sites on the bead are used up. In other embodiments,less than about 2%, less than about 5%, less than about 10%, less thanabout 20%, less than about 30%, less than about 40%, or less than about50% of the DNA barcode attachments sites on the bead are used up. Instill other embodiments, with attachment of the first DNA barcodemodule, between 2-4%, between 2-6%, between 2-8%, between 2-10%, between2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%,between 10-20%, between 10-25%, between 10-30%, between 10-35%, between10-40%, of the DNA barcode attachment sites are used up.

Regarding limits, with attaching the last of the DNA barcode modulesthat make up a particular DNA barcode, less than 20% of the sites areused up, less than 30%, less than 40%, less than 50%, less than 60%,less than 70%, less than 80%, less than 90%, less than 95%, or less than98% of the sites are used up.

Exclusionary embodiments can exclude beads or methods that match any ofthe above values or ranges. Also, exclusionary embodiments can excludebeads or methods that fail to match any of the above values or ranges.

The following concerns polymers that comprises one or more nucleicacids, each being a DNA barcode, as well as polymers that comprise twoor more nucleic acids, where some of the nucleic acids have abiochemical function such as serving as a primer-annealing site or as aspacer, and where other nucleic acids have an informational function andare DNA barcodes. In exclusionary embodiments, the present disclosurecan exclude a DNA barcode that includes a DNA crosslinking agent such aspsoralen. Also, what can be excluded is a DNA barcode with a primerbinding region with a higher melting temperature (or a lower meltingtemperature) than a DNA barcode module. This temperature can be merely“higher” or “lower” or it can be at least 2 degrees C. higher, at least4 degrees C. higher, at least 6 degrees C. higher, at least 8 degreeshigher, or at least 2 degrees C. lower, at least 4 degrees C. lower, atleast 6 degrees C. lower, at least 8 degrees lower.

Also what can be excluded is a method for making a DNA barcode that usesDNA ligase. Also, what can be excluded is a DNA barcode and methods formaking, that comprise a hairpin (ssDNA bent in a loop, so that oneportion of the ssDNA hybridizes to another portion of the same ssDNA).Additionally, what can be excluded is a composition with a nucleic acidhairpin, where the nucleic acid hairpin is covalently closed, forexample, with a chemical linker. Moreover, what can be excluded is a DNAbarcode that is covalently linked, either directly to a “headpiece,” orindirectly to “headpiece” (indirectly by way of covalent binding to oneor more chemicals that reside in between DNA barcode and the headpiece).

In other exclusionary embodiments, what can be excluded is a bead-boundDNA barcode, where the completed DNA barcode does not comprise anydouble stranded DNA (dsDNA), but only comprises single stranded DNA(ssDNA).

Extent of using up attachment sites on a given bead, with synthesis ofan orthogonal-configured bead (regarding the 2^(nd) DNA barcode). Thefollowing concerns attaching the second DNA barcode module. Inembodiments, with attachment of the second DNA barcode module (for thecreation of the orthogonal configured bead), about 5%, about 10%, about20%, about 30%, about 40%, or about 50% of the remaining free DNAbarcode attachments sites on the bead are used up. In other embodiments,less than about 5%, less than about 10%, less than about 20%, less thanabout 30%, less than about 40%, or less than about 50% of the remainingfree DNA barcode attachments sites on the bead are used up. In stillother embodiments, with attachment of the first DNA barcode module,between 2-4%, between 2-6%, between 2-8%, between 2-10%, between 2-12%,between 2-14%, between 2-16%, between 2-18%, between 2-20%, between10-20%, between 10-25%, between 10-30%, between 10-35%, between 10-40%,of the remaining free DNA barcode attachment sites are used up.

Exclusionary embodiments can exclude beads or methods that match any ofthe above values or ranges. Also, exclusionary embodiments can excludebeads or methods that fail to match any of the above values or ranges.

The above embodiments, as well as the above exclusionary embodiments,can also be applied to a method with attaching a third DNA modulebarcode, or with attaching a fourth DNA module barcode, or withattaching a fifth DNA barcode module, and so on.

Concatenated-style DNA barcode (all DNA barcode modules reside in onechain or polymer, where the entire chain or polymer is attached to onelocation on the bead).

Synthesis of bead-bound concatenated-style DNA barcode. The presentdisclosure provides a bead-bound concatenated-style DNA barcode, wherethe bead contains a plurality of concatenated-style DNA barcodes, andwhere most or nearly all of the plurality of concatenated-style DNAbarcodes have essentially the same structure. The concatenated-style DNAbarcode can contain one or more DNA barcode modules, where the orderingof these DNA barcode modules (from the bead-attachment terminus to thedistal terminus) along the entire DNA barcode, takes the same order asthe time that the bead-bound concatenated-style DNA barcode issynthesized. Also, the ordering of these DNA barcode modules along theentire DNA barcode, takes the same order as the time that acorresponding chemical library monomer is coupled to the growingbead-bound compound.

The concatenated-style DNA barcode can comprise, in this order, a linkerthat is used to couple the entire concatenated-style DNA barcode to thebead. Also, it can comprise, in this order, a 1^(st) DNA barcode module,a 1^(st) annealing site, a 2^(nd) DNA barcode module, a 2^(nd) annealingsite, a 3^(rd) DNA barcode module, and a 3^(rd) annealing site.

One ordering of sequencing primer hybridizing site in a bead-bound DNAbarcode. In sequencing primer hybridizing site embodiments, theconcatenated-style DNA barcode can comprise, in this order, a linker, a1^(st) DNA barcode module, a 1^(st) annealing site, a 1^(st) sequencingprimer binding site, a 2^(nd) DNA barcode module, a 2^(nd) annealingsite, a 2^(nd) sequencing primer binding site, a 3^(rd) DNA barcodemodule, a 3^(rd) annealing site, and a 3^(rd) sequencing primer bindingsite, and so on.

Another ordering of the sequencing primer hybridizing site, as it occursin a bead-bound DNA barcode. In another sequencing primer hybridizingsite embodiment, the concatenated-style DNA barcode can comprise, inthis order, a linker, a 1^(st) DNA barcode module, a 1^(st) sequencingprimer binding site, 1^(st) annealing site, a 2^(nd) DNA barcode module,a 2^(nd) sequencing primer binding site, a 2^(nd) annealing site, a3^(rd) DNA barcode module, a 3^(rd) sequencing primer binding site, and3^(rd) annealing site, and so on.

The term “annealing site.” The term “annealing site” is used to refer toan annealing site that is part of a splint oligonucleotide (splintoligo) and also to refer to the corresponding bead-bound annealing sitethat resides on a growing bead-bound DNA barcode. The skilled artisanunderstands that the “annealing site” on the splint oligo does notpossess the same DNA sequence as the corresponding “annealing site” onthe growing bead-bound DNA barcode. In other words, the skilled artisanunderstands that one sequence is complementary to the other sequence.Therefore, it is of no consequence that, for the descriptions herein,both annealing sites have the same name. In other words, it is of noconsequence that the 2^(nd) annealing site on a splint oligo isdisclosed as one that hybridizes to the 2^(nd) annealing site on growingbead-bound DNA barcode.

Synthesis in blocks. In an alternative embodiment, the growing compoundand the growing sequence of DNA barcode modules can be synthesized inblocks. For example, a block consisting of 2-chemical library units canbe attached to a bead in parallel with attaching a block consisting ofcorresponding 2-DNA barcode modules. Similarly, a block consisting of3-chemical library units, can be attached to a bead in parallel withattaching a block consisting of a corresponding 3-DNA barcodes. Blocksynthesis involving blocks of four, blocks of five, blocks of six,blocks of seven, blocks of eight, blocks of nine, blocks of ten, and soon, are also provided. Each of these block transfer embodiments can alsobe excluded by the present disclosure. The blockwise transfer of DNAbarcode monomers can be done orthogonally, with unique attachment pointsfor receiving each of successive blocks of DNA barcode monomers.Alternatively, blockwise transfer of DNA barcode monomers can be done toproduce a concatemer structure (all DNA barcode modules occurring asonly one continuous, linear polymer).

Also, during split-and-pool synthesis in parallel of the bead-bound DNAbarcode and the bead-bound compound, synthesis of can occur in blocks.The block can take the form of two or more chemical library monomers,and the block can take the form of two or more DNA barcode modules.

Location of split-and-pool synthesis. Split-and-pool synthesis can beused for the parallel synthesis of bead-bound compounds and bead-boundconcatenated DNA barcode. Also, split-and-pool synthesis can be used forthe parallel synthesis of bead-bound compounds and bead-bound orthogonalDNA barcode. The concatenated DNA barcode can be made by way of the“splint oligo” method. Alternatively, concatenated DNA barcode can bemade by way of click chemistry. Also, a combination of the “splintoligo” method and click chemistry can be used. Split-and-pool synthesiscan occur in a 96 well plate, where each well has a floor made of a 0.25micrometer filter. Under normal gravity conditions, aqueous solutions donot flow through this filter. However, suction can be applied to removeany aqueous solutions from all of the 96 wells, for example, where thereis a need to replace a first aqueous solution with a second aqueoussolution. This suction method is used when the bead is exposed to afirst set of reagents, or when the first set of reagents needs to berinsed out, or when the first set of reagents needs to be replaced by asecond set of reagents. A manifold is used to hold the 96 well plate(Resprep VM-96 manifold) and a pump can be used to draw fluid out thebottom of every filter (BUCHI Vac V-500 pump). The 96 well plate withthe filter bottom was, AcroPrep Advance 96 well, 350 uL, 0.45 um, REF8048 (Pall Corp., Multi-Well Plates, Ann Arbor, Mich.).

Distance from primer annealing site to a DNA barcode module. For thepurpose of sequencing a bead-bound DNA barcode, that is, for the goal ofsequencing all of the DNA barcode modules that form the DNA barcode, apolynucleotide comprising a first nucleic acid that is an annealing sitefor a sequencing primer, and a second nucleic acid that is a DNA barcodemodule, the first nucleic acid can be immediately upstream of the secondnucleic acid. Alternatively, the first nucleic acid can be upstream ofthe second nucleic acid, where the first and second nucleic acids areseparated from each other by one, two, three, four, five, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or more nucleotides, or by about one, about two,about three, about four, about five, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, or about 15nucleotides. The separation can be with nucleic acids that merely serveas a spacer or, alternatively, the separation can be with a thirdnucleic acid that encodes information, such as step number in amulti-step pathway of organic synthesis, or the name of a class ofchemical compounds, or a disease that might be treatable by thebead-bound compound, or the date, or a lot number, and so on.

Synthesis of Bead-Bound Concatenated DNA Barcode Using Click Chemistry

Click chemistry can be used for the step-by-step synthesis of a DNAbarcode. Here, what can be coupled is a first DNA barcode moduledirectly to a bead, or a first DNA barcode module to a bead-boundlinker.

Also, what can be coupled is a polynucleotide taking the form of a firstnucleic acid that is a 1^(st) DNA barcode module attached to a secondnucleic acid that is a 1^(st) sequencing primer binding site. Thissequencing primer binding site allows the operator to determine thesequence of the 1^(st) DNA barcode module.

To provide another example, what can be coupled is a 2^(nd) DNA barcodemodule directly to a bead-bound 1^(st) DNA barcode module.Alternatively, what can be coupled is a polynucleotide taking the formof a first nucleic acid that is a 2^(nd) DNA barcode module attached toa second nucleic acid that is a 2^(nd) sequencing primer binding site.This sequencing primer binding site allows the operator to determine thesequence of the 2^(nd) DNA barcode module. If there is read-through tothe 1^(st) DNA barcode module, then what can be determined is thesequence of both of these DNA barcode modules.

To provide yet another example, what can be coupled is a polynucleotidecomprising a first nucleic acid that is a 1^(st) DNA barcode module, anda second nucleic acid that identifies the step in a multi-step parallelsynthesis of the DNA barcode and of the compound. Also, oralternatively, the second nucleic acid can identify the general class ofcompounds that are being made by the split-and-pool synthesis. Also, oralternatively, the second nucleic acid can identify a disease that is tobe treated by the compounds to be screened. Also, the second nucleicacid can identify the date, or the name of the chemist, and so on.

A preferred method for synthesizing the DNA barcode is shown below,where the same cycle of reactions is used with progressive attachment ofeach DNA barcode module.

Step 1. Provide a bead with an attached TCO group. In actual practice,the bead will have hundreds or thousands of identically attached TCOgroups, where each TCO group is attached to a different site on thebead. Also, in actual practice, a large number of beads will besimultaneously modified by click chemistry, with employment of thesplit-and-pool method.

Step 2. Add [tetrazine]-[first DNA barcode module]-[azide] to the bead,and allow the TCO group condense with the tetrazine group. The result isthe following construct: BEAD-TCO-tetrazine-first DNA barcodemodule-azide. In actual practice, this construct does not include anyTCO or tetrazine, but instead has the condensation product that iscreated when TCO condenses with tetrazine.

Step 3. Optional wash.

Step 4. Add DBCO-TCO in order to cap the azide and to create a TCOterminus The result is the following structure:

BEAD-TCO-tetrazine-first DNA barcode module-azide-DBCO-TCO

Step 5. Optional wash.

Step 6. Add the following reagent, which attaches the second DNA barcodemodule. Attachment is to the distal terminus of the growing DNA barcode.The reagent is:

[tetrazine]-[second DNA barcode module]-[azide] to the bead, and allowthe TCO group condense with the tetrazine group. The result is thefollowing construct:

BEAD-TCO-tetrazine-first DNA barcodemodule-azide-DBCO-TCO-[tetrazine]-[second DNA barcode module]-[azide]

The above scheme includes a cycle of steps for the stepwise addition ofmore and more DNA barcode modules, where these additions are in parallelwith additions of more and more chemical monomers. As stated elsewhere,this “parallel” synthesis can involve attaching a chemical monomerfollowed by attaching a DNA barcode module that identifies that monomeror, alternatively, attaching a DNA barcode module followed by attachinga chemical monomer that is identified by that particular chemicalmonomer.

Compounds for Click-Chemistry Synthesis of DNA Barcode

FIG. 17 discloses the chemical synthesis of a compound suitable forconnecting a deoxycytidine reside (dC) during the synthesis of a DNAbarcode module and, ultimately, the entire DNA barcode. The startingmaterial is N4-acetyl-2′-deoxy-5′-O-DMT cytidine. The abbreviation “DMT”stands for 4,4-dimethoxytrityl. The final product of this multi-steppathway of organic synthesis bears a cytosine moiety, a triphosphategroup, and a propargyl group that is attached to the 3′-position of theribose group. The propargyl group is used for click chemistry, where itcondenses with an azide group to produce a covalent bond. Aftercondensing, the result is that a residual chemical (never naturallypresent in nucleic acids), occurs as a “scar” from the click chemistrythat had been performed. What is available is DNA polymerases that canbe used for sequencing-by-synthesis of DNA barcodes made by clickchemistry, and where the DNA polymerases can move across the scars, andwhere the scars do not cause sequencing errors. TBAI is tetrabutylammonium iodide.

Synthesis of Concatenated Configuration DNA Barcode

In the following description, DNA barcode modules are assembled in a rowin order to create the DNA barcode. However, in the in-text diagramsthat are shown below, the term “DNA barcode” is used instead of “DNAbarcode module,” in order to make the in-text diagrams fit on the page.FIG. 7 illustrates the same steps as shown here, but with additionaldetails, such as diagrams of beads. A reiterated sequence of reactionscan be used for adding each additional DNA barcode module.

Option of creating a DNA barcode that includes a terminal nucleic acidthat encodes DNA hairpin. This concerns a DNA barcode that includes, atthe 3-prime end, a nucleic acid that possesses an annealing site for asequencing primer, a bend taking the form of about four bases that arenot base-paired, and a sequencing primer that is capable of bendingaround and forming base pairs with the sequencing primer annealing site.To repeat, the sequencing primer anneals to the sequencing primerannealing site, where the actual sequencing reaction begins at the3′-terminus of the annealed sequencing primer.

When it is time to perform a final step in synthesizing a DNA barcode,and when the final DNA barcode module is to be coupled to the growingbead-bound DNA barcode, the “splint oligo” can include a sequence thatencompasses a DNA hairpin (the DNA hairpin including, in this order, anannealing site for the sequencing primer, several nucleotides that donot base pair with each other or with any nearby sequences of bases, anda sequencing primer). After annealing the “splint oligo,” then DNApolymerase and dNTPs are added, where polymerization occurs at the3′-end of the growing DNA barcode, where what gets polymerized using thesplint oligo as a template is, in order: (1) Annealing site forsequencing primer, (2) Bend in the hairpin taking the form of four orfive deoxyribonucleotides that do not base pair with teach other, and(3) Sequencing primer.

Reversible terminator group at the 3′-end of the hairpin sequencingprimer. The present disclosure provides reagents, compositions, andmethods, for attaching a pre-formed complex of a nucleotide/reversibleterminator group, to the 3′-terminus of the annealed sequencing primer.Reversible terminator group is an optional component of the hairpinsequencing primer, where it is to be part of a bead-bound DNA barcode.

STEP 1. At the start, we have a bead situated in a picowell, where thebead bears a coupled polynucleotide, and where the 5′-end of thepolynucleotide is coupled to the bead, optionally, with a linker. FIG. 7shows that the bead-bound polynucleotide comprises a 1^(st) DNA barcodeand a 1^(st) annealing site. The linker can be made of a nucleic acid,or it can be made of some other chemically. Preferrably, the linker ishydrophobic, and preferably the linker separates the bead-bound DNAbarcode from the hydrophobic polystyrene bead, for example, a TentaGel®bead.

For convenience in writing, a 1^(st) annealing site that is part of abead-bound DNA barcode and a 1^(st) annealing site that is part of asoluble “splint oligo” are both called “1^(st) annealing site,” eventhough they do not have the same sequence of bases (instead, thesequence of bases are complementary to each other, where the result isthat the splint oligo can hybridize to the 1^(st) annealing site on thebead-bound growing DNA barcode, thereby serving as a template for DNApolymerase to extend the bead-bound DNA barcode by copying what is onthe splint oligo.

Also, for convenience in writing, a 2^(nd) annealing site that is partof a bead-bound DNA barcode and a 2^(nd) annealing site that is part ofa soluble “splint oligo” are both called, “2^(nd) annealing site,” eventhough they do not have the same sequence (but instead havecomplementary bases).

The bead-bound growing DNA barcode, from the 5′-end to the 3′-end, maycontain the nucleic acids in the following order:

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/

Alternately, the bead-bound growing DNA barcode, from the 5′-end to the3′-end, may include a nucleic acid that encodes the step number, wherethe bead-bound growing DNA barcode has nucleic acids in the followingorder:

Bead-/1^(st) DNA Barcode/Nucleic Acid Encoding Step Number/1^(st)Annealing Site/

Alternatively, the bead-bound growing DNA barcode can include a nucleicacid that is a functional nucleic acid (a sequencing primer annealingsite), as shown below:

Bead-/1^(st) DNA Barcode/Sequencing Primer Annealing Site/1^(st)Annealing Site/

What is not shown in these in-text diagrams is an optional linker thatmediates coupling of the DNA barcode to the bead. The linker can takethe form of a nucleic acid, or it can be made of some other organicchemical.

STEP 2. Add a soluble splint oligonucleotide (splint oligo), where thissplint oligo comprises a 1^(st) annealing site and a 2^(nd) DNA barcodemodule, and a 2^(nd) annealing site.

FIG. 7 also illustrates the step where the hybridized splint oligo isused as a template, where DNA polymerase catalyzes the attachment to thebead-bound growing DNA barcode of the 2^(nd) DNA barcode module and the2^(nd) annealing site. FIG. 7 shows the enzymatic product where DNApolymerase catalyzes uses the splint oligo as a template, resulting inthe bead-bound DNA barcode growing by a bit longer (growing by covalentattachment of the 2^(nd) DNA barcode and the 2^(nd) annealing site. Whatis shown immediately below in the text, is the complex of the splintoligo that is hybridized to the bead-bound growing DNA barcode:

Bead-/1^(st) DNA barcode/1^(st) annealing site/

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

To reiterate some information shown in FIG. 7 , what is shownimmediately below is the splint oligo:

“1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site”

STEP 3. DNA polymerase and dNTPs are added to extend the bead-bound DNAbarcode. Shown below is the bead-bound growing DNA barcode, with thesplint oligo still hybridized to it, and where the bead-bound growingbarcode is longer than before, because what is now attached to it is anucleic acid that is the “2^(nd) DNA barcode module” and a nucleic acidthat is the “2^(nd) annealing site.” FIG. 7 also illustrates this step.The splint oligo is shown underneath the bead-bound growing barcode:

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd)Annealing Site

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

Step 4. Wash away the splint oligo. The splint oligo can be encouragedto dissociate from the bead-bound growing barcode by heating, that is,by heating the entire picowell plate, for example, to about 60 degreesC., about 65 degrees C., about 70 degrees C., about 75 degrees C., about80 degrees C., for about ten minutes or, alternatively, by adding diluteNaOH to the picowell array, and then neutralizing.

Step 5. Add a second splint oligo which, after hybridizing to thebead-bound growing splint oligo, can be used as a template for mediatingDNA polymerase-catalyzed attachment of a 3^(rd) DNA barcode and a 3^(rd)annealing site. This second splint oligo, which is a soluble reagent, isshown below (but it is not shown in FIG. 7 ):

2^(nd) Annealing Site/3^(rd) DNA Barcode/3^(rd) Annealing Site/

Step 6. Allow this oligonucleotide to anneal to the correspondingbead-bound “2^(nd) annealing site,” and allow DNA polymerase to extendthe bead-bound oligonucleotide, so that it contains a complement to the:“3^(rd) DNA barcode/3^(rd) annealing site/

Step 7. Wash away the second splint oligo.

Step 4. Add the following splint oligo (this particular addition is notshown in FIG. 7 ).

3rd Annealing Site/4th DNA Barcode/4th Annealing Site/

This soluble oligonucleotide has a nucleic acid that can anneal to the“3^(rd) annealing site” of the bead-bound oligonucleotide. Onceannealed, DNA polymerase with four dNTPs are employed and used forextending the bead-bound oligonucleotide to encode yet another DNAbarcode module (the 4^(th) DNA barcode). The above cycle of steps isrepeated, during the entire split-and-pool procedure that creates, inparallel, the library of chemical compounds and the associated DNAbarcodes, where each DNA barcode is associated with a given compound(where each DNA barcode informs us of the history of chemical synthesisof the associated compound). The above cycle of steps is stopped, whenthe chemical synthesis of the library of compounds has been completed.With the completed bead-bound, DNA barcoded chemical library in hand,the beads can then be dispensed into picowells of a picowell array.

The DNA barcode for each bead also constitutes a DNA barcode that isassociated with each picowell. The DNA barcode allows identification ofthe bead-bound compound. The sequencing method of the present disclosureoccurs inside the picowell while the bead is still inside the picowell.In exclusionary embodiments, the present disclosure can exclude anysequencing method and can exclude any reagents used for sequencing,where sequencing is not performed on a DNA template that is bead-bound,or where sequencing is not performed on a bead-bound DNA template thatis situated inside a picowell.

Annealing sites for sequencing primer. In one embodiment, each DNAbarcode module in a completed DNA barcode is operably linked and inframe with its own sequencing primer annealing site, thus providing theoperator with the ability to conduct separate sequencing procedures oneach DNA barcode module (in this embodiment, it is preferred that eachDNA barcode module is also operably linked with its own nucleic acidthat identifies (encodes) the step in synthesis of the entire DNAbarcode.

In another embodiment, each DNA barcode has only one sequencing primerannealing site, where this can be situated at or near the 3′-terminus ofthe bead-bound DNA barcode, and where the sequencing primer itself canbe soluble, added to the picowell, and then hybridized to the sequencingprimer annealing site. Alternatively, where the sequencing primer is tobe part of a DNA hairpin, this DNA hairpin is added by way of a “splintoligo” at the final step in creating the bead-bound DNA barcode. FIG. 7does not show any annealing sites for any sequencing primers.

Nucleic Acids Coupled to Beads by Way of the 3′-Terminus of the NucleicAcid

While various embodiments disclosed in this invention pertain tocoupling DNA to a bead by way of the DNA's 5′-end, in other embodiments,DNA such as a DNA barcode or a DNA tag, can be coupled to the bead byway of their 3′-end. The 3′-hydroxyl group of DNA might be reactiveunder certain chemical synthesis conditions (e.g. Mitsunobutransformations), rendering the 3′-end damaged and unable to participatein extension, ligation or other steps. Thus DNA tags may be attached tobeads via their 3′-ends to prevent unwanted chemical reactions and toprevent damage to the DNA barcodes.

Exclusionary embodiments regarding bead-bound DNA barcodes of thepresent disclosure. What can be excluded is any bead, microparticle,microsphere, resin, or polymeric composition of matter, wherein theconcatenated DNA barcode is linked to the bead by way of aphotocleavable linker or by way of a cleavable linker.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that does not include both of thefollowing: (1) Concatenated DNA barcode that is coupled to a firstposition on the bead, (2) A compound that is coupled to a secondposition on the bead, and wherein the first position is not the same asthe second position. In a preferred embodiment, this “compound” is madeof a plurality of chemical library monomers.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that does not have an exterior surface(or exterior surfaces) and also an interior surface (or interiorsurfaces, or interior regions), and where the bead does not comprise atleast 10,000 substantially identical concatenated DNA barcodes that arecoupled to the bead, and wherein at least 90% of the at least 10,000substantially identical concatenated DNA barcodes are coupled to theexterior surface. In other words, what can be excluded is any bead whereat least 90% of the coupled concatenated DNA barcodes are not coupled tothe exterior surface.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that is made substantially ofpolyacrylamide or that contains any polyacrylamide.

What can be excluded is any bead, microparticle, microsphere, hydrogel,resin, or polymeric composition of matter, that contains a promoter,such as a T7 promoter, or that contains a polyA region, or that containsa promoter and also a polyA region.

Method with only one cycle of annealing/polymerization, to produce abead-bound DNA barcode with two DNA barcode modules. The presentdisclosure encompasses systems, reagents, and methods, where thebead-bound DNA barcode includes only one annealing/polymerization step.This embodiment is represented by the following diagrams, where thefirst diagram shows annealing of the splint oligo, and the seconddiagram shows filling-in using DNA polymerase. The end-result is abead-bound DNA barcode that contains two DNA barcode modules. In thisparticular procedure, the bead-bound starting material can optionallyinclude linker (but preferably not any cleavable linker), optionally anucleic acid that encodes information other than identifying a chemicalcompound, and optionally a functional nucleic acid, such as a sequencingprimer or a DNA hairpin. The two diagrams are shown in the text (see,immediately below):

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/2^(nd) DNA Barcode/2^(st)Annealing Site

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

Method with two cycles of annealing/polymerization, to produce abead-bound DNA barcode that has three DNA barcode modules. The presentdisclosure encompasses bead-bound compositions, systems, and methods,where two different split oligos are used (first splint oligo, secondsplint oligo). In this situation, the first splint oligo comprises thestructure: 1^(st) annealing site/2^(nd) DNA barcode/2^(nd) annealingsite, and where the second splint oligo comprises the structure: 2^(nd)annealing site/3^(rd) DNA barcode/3^(rd) annealing site.

Method with three cycles of annealing/polymerization, to produce abead-bound DNA barcode that has four DNA barcode modules. The presentdisclosure encompasses bead-bound compositions, systems, and methods,where three different split oligos are used (first splint oligo, secondsplint oligo, third splint oligo). In this situation, the first splintoligo comprises the structure: 1^(st) annealing site/2^(nd) DNAbarcode/2^(nd) annealing site, and where the second splint oligocomprises the structure: 2^(nd) annealing site/3^(rd) DNA barcode/3^(rd)annealing site, and where the third splint oligo comprises thestructure: 3^(rd) annealing site/4^(th) DNA barcode/4^(th) annealingsite.

Method with four cycles of annealing/polymerization, to produce abead-bound DNA barcode that has five DNA barcode modules. The presentdisclosure encompasses bead-bound compositions, systems, and methods,where four different split oligos are used (first splint oligo, secondsplint oligo, third splint oligo, fourth splint oligo). In thissituation, the first splint oligo comprises the structure: 1^(st)annealing site/2^(nd) DNA barcode/2^(nd) annealing site, and where thesecond splint oligo comprises the structure: 2^(nd) annealingsite/3^(rd) DNA barcode/3^(rd) annealing site, and where the thirdsplint oligo comprises the structure: 3^(rd) annealing site/4^(th) DNAbarcode/4^(th) annealing site, and where the fourth splint oligocomprises the structure: 4^(th) annealing site/5^(th) DNA barcode/5^(th)annealing site,

Embodiments with a plurality of steps of annealing/polymerization, toproduce a bead-bound DNA barcode that has a plurality of DNA barcodemodules. The present disclosure encompasses bead-bound compositions,systems, and methods, relating to concatenated barcodes, that uses onlyone splint oligo (making a 2-module DNA barcode), that uses only twosplint oligos (making a 3-module DNA barcode), that uses only threesplint oligos (making a 4-module DNA barcode), that uses only foursplint oligos (making a 5-module DNA barcode), that uses only fivesplint oligos (making a 6-module DNA barcode), that uses only six splintoligos (making a 7-module DNA barcode), and so on.

What is encompassed is bead-bound compositions, systems, and methods,that uses at least one splint oligo, at least two splint oligos, atleast three splint oligos, at least four splint oligos, at least fivesplint oligos, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at last 13, at least 14, at least 20splint oligos, or less than 20, less than 15, less than 10, less than 8,less than 6, less than 4, less than 3, less than 2 splint oligos. Thesenumbers refer to the splint oligo itself, as well as to the number ofthe step of adding the splint oligo, and also to the numbering of theDNA module that is added to the growing bead-bound DNA barcode.

Reducing Damage to DNA Barcodes

Reducing damage by using orthogonal DNA barcodes (instead ofconcatenated DNA barcodes). One way to get oriented to the topic ofconcatenated DNA barcodes and orthogonal DNA barcodes, is to noteadvantages that one has over the other. An advantage of orthogonalbarcoding over concatenated barcoding, is as follows. With attachment ofeach monomer of a growing chemical compound, what is attached inparallel are chemical library monomers to create a chemical library, andDNA barcode modules to create a completed and full-length DNA barcode.

With concatenated barcoding, if attachment of any given module isimperfect (meaning, that not all of the attachments sites weresuccessfully coupled with a needed module), then the sequence of thecompleted barcode will not be correct. The statement “not be correct”means that imperfect coupling resulted in chunks that were missing,where the user had assumed that the completed product was a completedand correct DNA barcode. Here, the completed DNA barcode sequence willcontain a mistake, due to failure of attachment of all of the DNAmodules. In contrast, with orthogonal barcoding each individual DNAmodule gets covalently bound to its own unique attachment site on thebead. And where once a DNA module gets attached to a given site on thebead, no further DNA modules need to get coupled to the DNA modules thatare already coupled to the bead.

Reducing damage by using cross-linkers. The present disclosure providesreagents and methods for reducing damage to bead-bound DNA barcodes, andfor reducing damage to to partially synthesized bead-bound DNA barcodes.Each DNA barcode module, prior to attaching to a growing bead-bound DNAbarcode, can take the form of double stranded DNA (dsDNA), where thisdsDNA is treated with a DNA cross-linker such as mitomycin-C. Aftercompletion of the synthesis of the DNA barcode in its dsDNA form, thisdsDNA is converted to ssDNA. Conversion of dsDNA to ssDNA can beeffected where one of the DNA strands has a uracil (U) residue, andwhere cleavage of the DNA at the position of the uracil residue iscatalyzed by uracil-N-glycosidase (see, FIG. 5 of Ser. No. 62/562,905,filed Sep. 25, 2017. Ser. No. 62/562,905 is incorporated herein byreference in its entirety). The above refers to damage that is inflictedon the growing DNA barcode by reagents used to make the bead-boundchemical compound.

Reducing damage by using double stranded DNA (dsDNA) for making DNAbarcodes. Another method for reducing damage to bead-bound DNA barcodes,and for reducing damage to partially synthesized DNA barcodes, is bysynthesizing the DNA barcode in a double stranded DNA form, where eachof the DNA barcode modules that are being attached to each other takesthe form of dsDNA, and where each of the two strands is stabilized byway of a DNA headpiece. For eventual sequencing of the completed DNAbarcode, one of the strands is cleaved off from the DNA headpiece andremoved. The above refers to damage that is inflicted on the growing DNAbarcode by reagents used to make the bead-bound chemical compound (wherethis chemical compound is a member of the chemical library).

Reducing damage by including a hairpin. Yet another method for reducingdamage to bead-bound DNA barcodes, is to synthesize the DNA barcode in away that self-assembles to form a hairpin, and where this DNA barcodeself-assembles to that the first prong of the hairpin anneals to thesecond prong of the hairpin.

Where the DNA barcode being synthesized takes the form of doublestranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denaturethe DNA barcode. The above methods and reagents can preventdenaturation.

Reducing damage by using sealed ends of dsDNA. Another method forreducing damage to bead-bound DNA barcodes, and for reducing damage topartially synthesized DNA barcodes, is to use double stranded DNA(dsDNA) and to seal the ends of this dsDNA by way of 7-aza-dATP anddGTP.

Reducing damage by avoiding proteic solvents, avoiding strong acids andbasis, avoiding strong reducing agents and oxidants. The type ofchemistry that is compatible with the presence of deoxyribonucleic acids(DNA), whether bead-bound DNA or DNA that is not bead-bound, may requireabsence of proteic solvents, avoiding strong acidic conditions, avoidingstrong basis such as t-butyl lithium, avoiding strong reducing agentssuch as lithium aluminum hydride, avoiding reagents that react with DNAbases, such as some alkyl halides, and avoiding some oxidants (see, Lukand Sats (2014) DNA-Compatible Chemistry (Chapter 4) in A Handbook forDNA-Encoded Chemistry, 1^(st) ed. John Wiley and Sons, Inc.).

As stated elsewhere, the term “DNA barcode” can refer to apolynucleotide that identifies a chemical compound in its entiretywhile, in contrast, “DNA barcode module” can refer to only one of themonomers that make up the chemical compound.

Reducing damage to nucleic acids by using DNA-compatible chemistry. Satzet al, disclose various chemistries that are compatible with bead-boundnucleic acids (Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632,correction in Satz et al (2016) Bioconjugate Chem. 27:2580-2580).Although the descriptions in Satz et al, supra, concern chemicalreactions that are performed on DNA/chemical library member conjugates,the types of DNA-compatible chemistries that are described are alsorelevant, where the organic chemistry is to be performed on a bead thatcontains bead-bound compounds and bead-bound DNA.

DNA-compatible reactions for the formation of benzimidazole compounds,imidazolidinone compounds, quinazolinone compounds, isoindolinonecompounds, thiazole comopunds, and imidazopyridine compounds aredisclosed (see, Satz et al, Table 1, entries 1-6).

Moreover, DNA-compatible protecting groups are disclosed as including,alloc deprotection, BOC deprotection, t-butyl ester hydrolysis,methyl/ethyl ester hydrolysis, and nitro reduction with hydrazine andRaney nickel (see, Satz et al, Table 1, entries 7-11).

Furthermore, methods for coupling reagents to DNA are disclosed, wherethe coupling occurs with a functional group that is already attached tothe DNA. The methods include Suzuki coupling, an optimized procedure forthe Sonogashira coupling between an alkyne and an arylhalide, theconversion of aldehydes to alkynes usingdimethyl-1-diazo-2-oxopropylphosphonate, a new method for triazole cycloaddition directly from purified alkyne, an improved method for reactionof isocyanate building blocks with an amine functionalized DNA where theimproved reaction occurs with isocyanate reagent at pH 9.4 buffer (see,Satz, et al, Table 1, entries 12-15).

Additional methods for coupling reagents to DNA are disclosed, where thecoupling occurs to a functional group already attached to the DNA. Theseinclude a method where a primary amine is conjugated to DNA, anoptimized procedure to form DNA-conjugated thioureas, a method toalkylate secondary amines and the bis-alkylation of aliphatic primaryamides, monoalkylation of a primary amine DNA-conjugate, usinghetarylhalides as building blocks that can be reacted withamine-functionalized DNA-conjugate, and methods for Wittig reactions(see, Satz et al, Table 1, entries 16-20).

Reducing damaged DNA by way of DNA repair enzymes. Various proteins,including enzymes, DNA-damage binding proteins, and helicases, areavailable for repairing DNA damage. What is commercially available isDNA repair proteins that can repair oxidative damage, radiation-induceddamage, UV light-induced damage, damage from formaldehyde adducts, anddamage taking the form of alkyl group adducts. Glycoside enzyme, whichremove damaged bases (but do not cleave ssDNA or dsDNA) are available torepair 5-formyluracil, deoxyuridine, and 5-hydroxymethyluracil. T4PDG isavailable to repair pyrimidine dimers. hNEIL1 as well as Fpg areavailable to repair oxidized pyrimidines, oxidized purines, apurinicsites, and apyrimidinic sites. EndoVIII is available to repair oxidizedpyrimidine and apyrimidinic sites. EndoV is available for repairingmismatches. HaaG is a glycosylase that is available for repairingalkylated purines. Where a DNA repair enzyme leaves a gap, where doublestranded DNA has a gap where one or more continuous deoxyribonucleotidesare missing in one of the strands, then various DNA polymerases areavailable for filling in the gap (see, Catalog (2018) New EnglandBioLabs, Ipswich, Mass.).

A variety of DNA repair enzymes and DNA repair systems have beenisolated from mammals, yeast, and bacteria. These include those thatmediate nucleotide excision repair (NER), direct repair, base excisionrepair, transcription-coupled DNA repair, and recombinational repair.Interstrand DNA crosslinks can be repaired by combined use of NER andhomologous recombination. Direct repair includes repair of cyclobutanepyrimidine dimers and 6-4 products, by way of photolyase enzymes. Directrepair also includes removal of O⁶-methyl from O⁶-methylguanine by DNAmethyltarnsferase. See, Sancar et al (2004) Ann. Rev. Biochem. 73:39-85,Hu, Sancar (2017) J. Biol. Chem. 292:15588-15597.

The present disclosure provides systems, reagents, and methods forrepairing damage to bead-bound DNA barcodes by treating with a DNArepair enzyme, or by a complex of DNA repair proteins, and the like.

Reducing damage via coupling DNA to beads via their 3′-end. Certainchemical transformation may damage exposed 3′-hydroxyl groups of nucleicacids. For instance Mitsunobu reactions allow the conversion of primaryand secondary alcohols to esters, phenyl ethers, thioethers and variousother compounds, which might render exposed 3′-ends unreactive tosubsequent processing steps, or cause the now modified 3′-end toparticipate in further chemical reactions. In some embodiments, the DNAtags may be attached to beads via their 3′-end, so only the 5′-end isexposed to solution.

The reagents, systems, and methods of the present disclosure encompassbead-bound nucleic acids, such as a bead-bound DNA or a bead-bound DNAtags, where coupling to the bead involves the 3′-terminus (or the3′-end) of the DNA. Where ssDNA that comprises a DNA barcode is coupledby way of the 3′-end, of the ssDNA, sequencing can be initiated byhybridizing only one sequencing primer, where this sequencing primerhybridizes upstream of the entire DNA barcode, and where thishybridizing is at or near the bead-bound end of the coupled ssDNA. As analternative to using only one sequencing primer, a plurality ofsequencing primers can be used, where each sequencing primer hybridizesupstream to a particular DNA barcode module. For example, if a given DNAbarcode contains five DNA barcode modules, and where the DNA is coupledto a bead by way of its 3′-end, the DNA barcode can include fivedifferent primer annealing sites, where each primer annealing site islocated just upstream, or immediately upstream, of a given DNA barcodemodule.

Double stranded DNA (dsDNA) coupling embodiments. In other embodiments,what is coupled to the bead is dsDNA, where the 3′-terminus of only oneof the strands in the dsDNA are coupled to the bead. In a 5′-couplingembodiment that involves dsDNA, what can be coupled is dsDNA, where the5′-terminus of only one of the strands of the dsDNA is coupled to thebead.

(V) Coupling Chemical Compounds to Beads

The present disclosure provides: (1) Linkers to attach chemical librarymember to a substrate, such as a bead, (2) Linkers to attach nucleicacid barcode to a substrate, such as a bead, (3) Cleavable linkers, forexample, cleavable by UV light, cleavable by an enzyme such as aprotease, (4) Non-cleavable linkers, (5) Bifunctional linkers, (6)Multi-functional linkers, and (7) Plurality of beads used for linking.Available, for example, is 4-hydroxymethyl benzoic acid (HMBA) linker,4-hydroxymethylphenylacetic acid linker (see, Camperi, Marani, Cascone(2005) Tetrahedron Letters. 46:1561-1564).

A “non-cleavable linker” may be characterized as a linker that cannot bedetectably cleaved by any reagent, condition, or environment, that isused during the steps of a given organic chemistry procedure.Alternatively, a “non-cleavable linker” may be characterized as a linkerthat cannot be cleaved, except by a reagent, condition, or environmentthat is unacceptably destructive towards other reactants, products, orreagents of a given organic chemistry procedure.

A bifunctional linker, or other multifunctional linker, can take theform of a fork (fork used by humans for consuming food), where thehandle of the fork is attached to a bead, and where each tine of thefork are linked to one of a variety of chemicals. For example, one tinecan be linked to a chemical library member. Another tine can be linkedto a DNA barcode. Yet another tine of the fork can be linked to a metalion.

Regarding use of a multiplicity of beads, the present disclosureprovides multiple-bead embodiments, such as: (1) A first bead containingattached nucleic acid barcode linked to a second bead, where the secondbead contains attached chemical library member, (2) A first beadcontaining an attached nucleic acid barcode linked to a second bead,where the second bead contains an attached chemical library member, andwhere a third bead is attached (to one or both of the first bead andsecond bead), and where the third bead contains a covalently attachedreagent. The attached reagent can be an enzyme, where the enzyme is usedfor assaying activity of the attached chemical library member.

(VI) Coupling Monomers Together to Make a Compound

Exemplary chemical monomers. Amino acid derivatives suitable for use aschemical monomers for the compositions and methods of the presentdisclosure are shown in FIG. 4 . The figure indicates a source of thechemicals, for example, AnaSpec EGT Group, Fremont, Calif.,Sigman-Aldrich, St. Louis, Mo., Acros Organics (part of ThermoFisherScientific), or Combi-Blocks, San Diego, Calif.

Additional chemical monomers are shown in FIGS. 22-27 . Each of FIGS.22-27 provides the structure, chemical name, and an associated DNAmodule barcode. As disclosed on the figures, compounds 1-6 (FIG. 22 ),the respective barcodes are ACGT, ACTC, AGAC, AGCG, AGTA, and ATAT. Forcompounds 7-10 (FIG. 23 ), the respective barcodes are, ATGA, CACG,CAGC, and CATA. For compounds 11-16 (FIG. 24 ), the respective barcodesare, CGAG, CGCT, CGTC, CTAC, CTGT, and GACT. For compounds 17-21 (FIG.25 ), the respective barcodes are GAGA, GCAC, GCTG, GTAG, and GTCA. Forcompounds 22-26 (FIG. 26 ), the respective barcodes are GTGC, TAGT,TATC, TCAG, and TCGC. And for compounds 27-30 (FIG. 27 ), the respectivebarcodes are TCTA, TGAT, TGCA, and TGTG. These barcodes are onlyexemplary. For any given library of compounds, a different collection ofDNA barcodes may be used to identify each of the chemical monomers thatare used to build the compounds in that library.

Coupling reactions. The following describes coupling chemical monomersto the bead and to each other, that is, where a first step is couplingthe first chemical monomer directly to the bead by way of a cleavablelinker, and where subsequent chemical monomers are then connected toeach other, one by one. The conditions disclosed below are DNAcompatible.

This describes methods to make three amino acid compounds on Tentagel®beads. The Fmoc protected resin (1 mg, Rapp polymere GmbH, 10 um,TentaGel M-NH2, 0.23 mmol/g) modified with Fmoc-Photo-Linker,4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or another appropriatelinker with Fmoc protection was suspended inside each well of a reactorplate (Merck Millipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL).The solvent was removed by application of a vacuum to the bottom of theplate with a Resprep VM-96 vacuum manifold. The Fmoc protecting groupwas removed by suspending the resin in 150 uL of a mixture of 5%piperazine, 2% DBU in DMF. The plate was sealed with an Excel ScientificAlumna Seal and shaken at 40 C for 15 min. The solvent was removed by anapplied vacuum and the deprotection procedure repeated for 5 min. Afterfiltration each well was washed with 150 uL each of 2×DMA, 3×DCM, 1×DMAwith a vacuum applied between each wash to remove the solvent. Each wellof resin was then acylated by the appropriate amino acid by adding 150uL of a pre-activated mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200mM DIC and 80 mM 2,4,6-trimethylpyridine that was allowed to sit for 2min at room temperature. The plate was again sealed and shaken for 1 hrat 40 degrees C. After filtration each well was washed with 150 uL eachof 2×DMA and 3×DCM. The beads in each well were re-suspended in 150 ulof DCM and each well's contents combined through pipetting into a singlereceptacle. The combined beads are thoroughly mixed and redistributedinto the plate through pipetting equal amounts in the appropriate wells(1 mg/well). The solvent was removed by an applied vacuum and each wellwas ready for the next appropriate step. For each additional amino acidcoupling, first the Fmoc deprotection step is repeated followed by thecoupling step with the desired amino acid. If a split and pool isrequired, the combining and redistribution method is repeated.

This describes a method for creating 3-mer amino acid by split-poolmethod on beads. The Fmoc protected resin (1 mg, Rapp polymere GmbH, 10um, TentaGel M-NH₂, 0.23 mmol/g) modified with Fmoc-Photo-Linker,4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or any other appropriatelinker was suspended inside each well of a reactor plate (MerckMillipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL). The solventwas removed by application of a vacuum to the bottom of the plate with aResprep® VM-96 vacuum manifold. The Fmoc protecting group was removed bysuspending the resin in 150 uL of a mixture of 5% piperazine, 2% DBU inDMF. The plate was sealed with an Excel Scientific Alumna Seal andshaken at 40 C for 15 min. The solvent was removed by an applied vacuumand the deprotection procedure repeated for 5 min. After filtration eachwell was washed with 150 uL each of 2×DMA, 3×DCM, 1×DMA with a vacuumapplied between each wash to remove the solvent. Each well of resin wasthen acylated by the appropriate AA by adding 150 uL of a pre-activatedmixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC and 80 mM2,4,6-trimethylpyridine that was allowed to sit for 2 min at roomtemperature. The plate was again sealed and shaken for 1 hr at 40 C.After filtration each well was washed with 150 uL each of 2×DMA, 3×DCM,1×DMA. For each additional AA coupling, first the Fmoc deprotection stepis repeated followed by the coupling step with the desired AA. Toanalyze each successive coupling, a 1 mg portion of beads was suspendedin 100 uL DMSO and exposed to full power of the 365 nm LED for twohours. The resin is filtered off and the filtrate injected onto anAgilent 1100 series LCMS equipped with a Agilent Poroshell SB-C-18,3.0×50 mm, 2.7 um column. A gradient of 5% CH3CN in 0.1% TFA in water to100 CH3CN in 0.1% TFA over 4 min at a flow rate of 1.2 mL/min andmonitored at 220 nm was ran.

Experiment to make non-amino acid pendant with lenalidomide (Revlimid®)attached. This would be attached to the last amino acid afterdeprotection. This was also done in a spin. Each well of resin wasacylated (after an Fmoc deprotection) with 150 uL of a 5 min preagedmixture of 40 mM chloro acetic acid, 40 mM Oxyma, 80 mM DIC, and 40 mMTMP in DMA. The plate was sealed and shaken at 40 C for 1 hr. Each wellwas washed with 150 uL each of 3×DMA, 3×DCM, and 2×DMA. The resin wasthen re-suspended in a suspension of 100 mM K2CO3 and 100 mM Rev in DMA.The plate was sealed and shaken for 3 hrs at rt. The resin was washedwith 150 uL each of 2×50/50 DMA/water, 3×DMA, 3×DCM, and 2×DMA.

Defining the degree of fidelity of synthesis of a chemical compound thatis attached to a given bead. This concerns the completed chemicalcompound, where the chemical compound is a member of a chemical library.Each chemical compound may be made, in part, or in full, from chemicalmonomers. The following characterizes chemical compounds that areattached to a given bead. This given bead may be the product ofsplit-and-pool based synthesis of a library of chemical compounds, whereeach bead possesses a unique chemical compound.

Members of a chemical library can be synthesized on a solid support,such as on a bead, by way of solid phase synthesis. Solid phasesynthesis of chemicals with peptide bonds is characterized by use of onethe following two chemical groups. The first chemical group is,N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, base labile). The secondchemical group is, tert-butyloxycarbonyl (tBoc, acid labile) (see,Vagner, Barany, Lam (1996) Proc. Natl. Acad. Sci. 93:8194-8199). Fmocand tBoc are protecting groups that can be used to protect peptidesubstrates, where the Fmoc group or tBoc group is attached to thealpha-amino group (Sigler, Fuller, Verlander (1983) Biopolymers.22:2157-2162).

Preferably, at least 99.5%, at least 99.0%, at least 95%, at least 90%,at least 85%, or at least 80% of the member of the chemical librarybound to a given bead, following completed synthesis, has exactly thesame chemical structure. It is possible that incomplete coupling thatmight occur at one or more steps in the multi-step synthesis of thechemical library member. For this reason, the compositions of thepresent disclosure may be characterized and limited by one of thefollowing limits or ranges.

What is also provided by the present disclosure are methods and reagentswhere at least 5%, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 95%, or at least 99%, of the members of thechemical library bound to a given bead has, following completedsynthesis, exactly the same chemical structure (these numbers do takeinto account, and reflect, errors that might occur during solid phasesynthesis, for example, failure of one growing compound to receive oneof the chemical monomers. Also, these numbers do take into account, andreflect, chemical damage to any of the monomers that might occur duringsolid phase synthesis).

In exclusionary embodiments, the present disclosure can exclude anymethod or reagent that does not meet one of the above cut-off values for“exactly the same structure.”

In an alternate embodiment, two beads, 3 beads, 4 beads, 5 beads, about5-10 beads, about 10-20 beads, about 20-40 beads, about 40-80 beads, ina population of beads, contain the same and identical chemical compound(without taking into account any errors in incorporation of chemicalmonomers during solid phase synthesis, and without taking into accountany chemical damage that occurs to a chemical monomer during organicsynthesis).

Introduction to click chemistry. According to Jewett et al, “Clickreactions are defined . . . as those that . . . [are] selective, highyielding, and having good reaction kinetics. A subclass of clickreactions whose components are inert to the surrounding biologicalmilieu is termed biorthogonal” (Jewett and Bertozzi (2010) Chem. Soc.Rev. 39:1272-1279). “Click chemistry” can be used for joining smallunits together with heteroatom links, such as carbon-X-carbon. Clickchemistry can be used alone, or in conjunction with other types ofchemical reactions, for the synthesis of drugs or drug candidates. Clickchemistry works well with procedures used for combinatorial chemistry.Reactions in click chemistry are characterized by high yields, by beingirreversible, by insensitivity to oxygen or water. Classes of chemicalreactions used in “click chemistry” include: (1) Cycloadditionreactions, especially from the 1,3-dipolar family and from hetero-DielsAlder reactions,

(2) Nucleophilic ring-opening reactions, as with strained heterocyclicmolecules such as epoxides, aziridines, and cyclic sulfates, (4)Carbonyl chemistry of the non-aldol type, and(5) Addition to carbon-carbon multiple bonds, as with oxidationreactions and some Michael addition reactions. Click chemistry reactionsare distinguished by their high thermodynamic driving force, oftengreater than 20 kcal/mol while, in contrast, non-click chemistryreactions involve forming bonds with only a modest thermodynamic drivingforce (Kolb and Sharpless (2003) Drug Discovery Today. 8:1128-1137,Kolb, Finn, Sharpless (2001) Angew. Chem. Int. Ed. 40:2004-2021).

Tetrazine and trans-cyclooctene (TCO). Tetrazine, such as,1,2,4,5-tetrazine, can react with trans-cyclooctene (TCO) by way of aDiels-Alder cyclo addition (Devaraj, Haun, Weissleder (2009) Angew.Chem. Intl. 48:7013-7016).

Hartig-Buchwald amination. Hartwig-Buchwald amination reactions can beused in the solid-phase synthesis of pharmaceuticals. This aminationreactions is used to synthesize carbon-nitrogen bonds, where thereaction involves: aryl-halide plus amine (R₁—NH—R₂), as catalyzed bypalladium, to produce an aryl product where the amine replaces thehalide, and where the nitrogen of the amino group is directly attachedto the aromatic ring. The end-result is a product involving a carbon (ofaryl group) to nitrogen (of amino group) bond. Stated another way, thereaction converts arylhalides into the corresponding anilines.Hartwig-Buchwald amination is compatible with a variety of amines, andis well-suited for combinatorial chemistry (Zimmermann and Brase (2007)J. Comb. Chem. 9:1114-1137).

Huisgen cycloadditions. Huisgen 1,3-dipolar cycloaddition reactionsinvolve alkynes and organic azides. Alkynes have the structure, R—C≡CH.Azides have the structure, R—N⁺═N═N⁻. Copper catalysts accelerate therate of the Huisgen cycloaddition reaction. The Huisgen reactionoperates by way of “click chemistry” or “click reactions.” Huisgenreaction, when catalyzed by copper, can produce a 1,2,3-triazole nucleussuitable for making small molecule drugs. Huisgen reaction is compatiblewith the presence of amino acid side chains, at least when in aprotected form. Molecules made with a 1,2,3-triazole may possess a bondthat is similar to the amide bonds of polypeptides, and thus thesemolecules can be a surrogate for the peptide bond (Angell and Burgess(2007) Chem. Soc. Rev. 36:1674-1689).

Peptide nucleic acids (PNAs). The present disclosure provides themethods of split and pool chemistry, combinatorial chemistry, or solidphase chemistry, for synthesizing peptide nucleic acids. Peptide nucleicacids are analogues of oligonucleotides. They resist hydrolysis bynucleases. They can bind strongly to their target RNA sequences. Uptakeof peptide nucleic acids into cells can be enhanced by “cell penetratingpeptides” (Turner, Ivanova, Gait (2005) Nucleic Acids Res. 33:6837-6849,Koppelhus (2008) Bioconjug. Chem. 19:1526-1534). Peptide nucleic acidscan be made by solid phase synthesis and by combinatorial synthesis(see, Quijano, Bahal, Glazer (2017) Yale J. Biology Medicine.90:583-598, Domling (2006) Nucleosides Nucleotides. 17:1667-1670).

The present disclosure encompasses bead-bound compounds, where thecompound takes the form of only one monomer. For example, thisbead-bound compound can take the form of lenalidomide, or it can takethe form of lenalidomide with an attached carboxylic acid group, or aform of lenalidomide where the amino group has been modified with asmall chemical moiety that bears a carboxylic acid group, or where thecompound is a lenalidomide analog that is a stereoisomer or anenantiomer of lenalidomide.

(VII) Split and Pool Synthesis and Parallel Synthesis

This concerns use of the “split and pool” method for synthesizing alibrary of chemical compounds, and the method where the “split and“pool” method is used for the simultaneous synthesis of bead-boundchemical compounds and bead-bound DNA barcodes. This also describessplitting and pooling to make a mixed set of compounds. At a laterpoint, what is disclosed below is coupling of a non-amino acid, as wellas the preparations of beads that are modified by polyethylene glycol(PEG).

The present disclosure provides split and pool synthesis for generatingchemical libraries. In one embodiment, this method involves the steps:(a) Split beads into different containers, (b) Add a different buildingblock to each container. For example, where three container are used,add and react Species A to the first containing, Species B to the secondcontainer, and Species C to the third container, where the speciesbecome covalently bound to attachment sites on whatever bead is in thecontainer, (c) Pool all beads together in one container, (d) Split beadsinto three containers, (e) Add a different building block to eachcontainer, where Species A is added to the first container, Species B isadded to the second container, and Species C is added to the thirdcontainer, where the species become covalently bound to the firstspecies that had been previously attached (see, Stockwell (2000) TrendsBiotechnol. 18:449-455).

The split-and-pool synthesis of the present disclosure includes, eitherbefore or after each chemical coupling step (making the chemical librarymember), a DNA-barcode coupling step, where this DNA barcode identifiesthe chemical that is being coupled in that step.

In exclusionary embodiments, the present disclosure can exclude methodsand reagents where, for a given step of parallel synthesis, a barcode isattached prior to attaching a chemical. Conversely, the presentdisclosure can exclude methods and reagents where, for a given step ofparallel synthesis, a chemical is attached prior to attaching a barcode.

One characteristics of a bead-bound chemical library that is prepared bythe split and pool method, is that each bead will have only one type ofcompound attached to it. Where there is incomplete coupling, forexample, if for a given split and pool step, only 4,000 out of 5,000attachment sites was successfully coupled with the desired chemicalspecies, then some heterogeneity will occur.

Parallel synthesis. In a preferred embodiment of the present disclosure,parallel synthesis can be used for organic synthesis of a chemicalcompound and of the associated DNA barcode. In actual practice,modification of a bead by one more chemical monomers and modification ofthe same bead by one more DNA barcode modules, is not strictly inparallel. In actual practice, the bead receives one more chemical unit(chemical monomer) followed by receiving a DNA barcode module thatencodes that particular chemical unit. The term “parallel” refers to thefact that, as the polymer of chemical library monomers grows, thepolymer of DNA barcode module also grows. When all of the DNA barcodemodules have been attached to the bead, to form either a CONCATENATEDstructure or an ORTHOGONAL structure, the full-length DNA barcode iscalled a “DNA barcode” (and not merely a DNA barcode module).

Ratio of number of externally attached DNA barcode to total number ofattached chemical library member.

This concerns external surfaces and internal surfaces of a bead. For agiven bead that has externally attached DNA barcodes (without regard tonumber of internally attached DNA barcodes) and attached chemicallibrary member (attached to both external surface as well as to internalsurfaces), the ratio of number of externally attached DNA barcode numbertotal attached chemical library member number can be, for example, about0.1:100, about 0.2:100, about 0.5:100, about 1.0:100, about 2:100, about5:100, about 10:100, about 20:100, about 30:100, about 40:100, about50:100, about 60:100, about 70:100, about 80:100, about 90:100, about1:1, about 100:150, about 100:200, about 100:400, about 100:600, and thelike. In exclusionary embodiments, the present disclosure can excludeany bead, or any population of beads, that fits into one of the abovevalues.

Homogeneity of DNA barcode for a typical bead, homogeneity of chemicallibrary member for a typical bead

The present disclosure provides, for any given bead (or for anypopulation of beads) a “chemical library homogeneity” that is at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least92%, at least 94%, at least 96%, at least 98%, at least 99.5%, and thelike.

In less stringent embodiment, the present disclosure provides, for anygiven bead or, alternatively, for any given population of beads, a“chemical library homogeneity that is at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50%.

Similarly, the present disclosure provides the above cut-off values forassessing homogeneity of a barcode, such as a DNA barcode.

Homogeneity for DNA barcode and homogeneity for a chemical librarymember may be defined, in terms, of percent of total population thatconforms to the exact sequence as planned and desired by the methodssection of a lab manual or notebook.

In exclusionary embodiments, the present disclosure can exclude anyreagent, composition, or method, that does not conform with one or moreof the above cut-off values.

Where one assesses homogeneity of a population of beads, one needs toaccount homogeneity for the sum of bead #1, bead #2, bead #3, bead #4,bead #5, bead #6, bead #7, and so on, for the situation wherehomogeneity is desired throughout the entire population of beads.

In exclusionary embodiments, the present disclosure can exclude anybead, or any population of beads, where homogeneity of DNA barcode isnot at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99.5%, and the like. Also, in exclusionary embodiments, the presentdisclosure can exclude any bead, or any population of beads, wherehomogeneity of chemical library member is not at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99.5%, and the like.

Ratio of internally attached DNA barcodes to externally attached DNAbarcodes

In some embodiments of the present disclosure, it might be desired tomanufacture and use beads where DNA barcodes are mainly attached on theexterior surface. One reason to NOT make and use beads with internal DNAbarcodes, is the low permeation of DNA oligomers to the interior spaces,and low permeation of DNA ligases to interior spaces (ligases forconnecting DNA modules to each other to create the finished DNAbarcode). And for sequencing purposes, a reason to NOT make and useinternal DNA barcodes, is low permeation of enzymes needed to amplifyDNA needed for eventual sequencing of the barcode. Yet another reasonNOT to make and use beads with internal DNA barcodes is to fee upinterior space for attaching members of the chemical library.

The present disclosure provides beads bearing DNA barcodes, where theratio of internally attached DNA barcodes to externally attached DNAbarcodes is about 0.1:100, about 0.2:100, about 0.4:100, about 0.8:100,about 1:100, about 2:100, about 4:100, about 8:100, about 10:100, about20:100, about 40:100, about 50:100, about 60:100, about 70:100, about80:100, about 90:100, about 1:1, and so on.

Also, the present disclosure provides beads bearing DNA barcodes, wherethe ratio of internally attached DNA barcodes to externally attached DNAbarcodes is under 0.1:100, under 0.2:100, under 0.4:100, under 0.8:100,under 1:100, under 2:100, under 4:100, under 8:100, under 10:100, under20:100, under 40:100, under 50:100, under 60:100, under 70:100, under80:100, under 90:100, under 1:1, and so on.

A population of beads, in an aqueous suspension, can be contacted to asubstrate, such as a picowell array, resulting in beads entering andoccupying the picowells. The ratio of the number of beads in thesuspension to the number of picowells in the substrate can be adjusted,to arrive at a desired occupancy. For example, if the suspensioncontains only one bead, then every picowell that contains a bead willcontain only one bead, where the remaining picowells will not containany bead. If the suspension contains 20,000 beads and if the substratecontains 200,000 picowells, then at least 180,000 picowells will betotally empty of beads, and where most of the picowells that contain abead will contain only one bead. A small percentage of occupiedpicowells will contain two beads.

In value embodiments, the ratio of bead number in the suspension topicowell number can be about 0.2:100, about 0.4:100, about 0.6:100,about 0.8:100, about 1:100, about 2:100, about 4:100, about 6:100, about8:100, about 10:100, about 20:100, about 30:100, about 40:100, about50:100, about 60:100, 80:100, about 100:100 (same as 1:1), about 2:1,about 4:1, about 6:1, about 8:1, about 10:1, and the like.

In exclusionary embodiments, the present disclosure can exclude anymethod or system, that falls into one of the above values or ranges.

In range embodiments, the ratio of bead number in the suspension topicowell number can be about 0.2:100 to about 0.4:100, about 0.4:100 toabout 0.6:100, about 0.6:100 to about 0.8:100, about 0.6:100 to about1:100, about 1:100 to about 2:100, about 2:100 to about 4:100, about4:100 to about 6:100, about 0.6:100 to about 8:100, about 8:100 to about10:100, about 10:100 to about 20:100, about 20:100 to about 30:100,about 30:100 to about 40:100, about 40:100 to about 50:100, about 50:100to about 60:100, about 60:100 to 80:100, about 80:100 to about 100:100(same as 1:1), about 100:100 (same as 1:1) to about 2:1, about 2:1 toabout 4:1, about 4:1 to about 6:1, about 6:1 to about 8:1, about 8:1 toabout 10:1, and the like.

In exclusionary embodiments, the present disclosure can exclude anymethod or system, that falls into one of the above values or ranges.

(VIII) Fabricating Picowells

Combination of UV light, photomask, and photoresist for manufacturing apicowell array plate. Plates that include many microwells or picowellscan be fabricated as follows for use in the present disclosure. Inbrief, a sandwich of three layers is assembled. The top layer isphotoresist. The middle layer is a glass wafer. The bottom layer is aphotomask. The picowells will be carved out of the photoresist by UVlight. After the picowells are carved out of the flat sheet ofphotoresist, the photoresist resembles a typical metal pan that containscups for baking muffins, and where the cups in the pan that are used forholding muffin batter have angled sides. The UV light acts as an“un-cross linker” because it breaks down the photoresist's polymer.After UV treatment, solvent is added to wash out the UV treatedphotoresist, leaving clean-looking picowells.

Rotating at an angle to create angled walls. Picowells with angled wallsare created as follows. The photomask has many apertures, where eachaperture corresponds to the desired bottom dimension of the picowell.The bottom dimensions can include a circumference, diameter, and ashape, that is, a round shape. The top dimension of the well is createdby directing an angled UV light towards the apertures in the photomaskwhile rotating the light source or rotating the stage that holds thesandwich (photomask/glass wafer/photoresist sandwich). With rotation,the light source is not at a 90 degree angle to thephotomask/wafer/photoresist sandwich, but instead is slightly angledaway from the 90 degree point, in order to carve out angled walls ineach picowell. The resulting picowell array plate that contains manypicowells can be used as is. Alternatively, this picowell array platecan be used as a mold for the inexpensive creating of many picowellarray plates.

Han et al describes equipment and reagents for manufacturing microwellplates where the microwells have angled walls (see, Han et al (2002) J.Semiconductor Technology and Science. 2:268-272). What is described is aUV source, a contact stage, a tilting stage, and the SU-8 photoresist.Fabrication begins with a single side polished silicon wafer. SU-8photoresist is coated on the wafer at about 0.10 to 0.15 mm thick. Then,the photoresist is soft baked on a 65 degrees C. hot plate for 10 minand then on a 95 degrees C. hot plate for 30 minutes. The resultingphotoresist/wafer sandwich is contacted with a UV mask using a contactstage. The term “Inclined and rotated UV lithography” refers to a methodfor manufacturing microwell array plates or picowell array plates, whereeach well has an angled wall. Here, the floor of the well has a smallerdiameter and the top of the well (where the top edge of the well meetsthe flat surface of the plate) has a wider diameter. For exposure withUV light, a turntable is used and where the UV light is inclined (Han etal, supra). The mask is contacted with the photoresist where each of theapertures in the mask are circular. FIG. 8 of Han et al, supra, providesa picture of the direction of UV light, the UV mask, the photoresiststructure, the wafer substrate, and the turntable. Han et al describeshow to manufacture a truncated cone. A soft material such as PDMS(polydimethylsiloxane) may be poured over the cone array and cured,whereupon peeling the PDMS layer, conical wells are formed.

Creating a mold for use in mass-production of picowell array plates.Where a picowell array plate has been manufactured, epoxy can be pouredover the plate resulting in filling all of the picowells and connectingall of the filled picowells with a platform of epoxy. Once the epoxy hassolidified, the solid platform with the attached array ofpicoprotuberances is removed (the picoprotuberance being the reverse ofthe desired picowell). The solid platform with picoprotuberances is areusable molding that can be used for the manufacture of many picowellarray plates.

The procedure for making replicates from the epoxy mold (or a cone arraymold made of any hard material is called, “hot embossing.” Briefly, asubstrate material is heated to its glass transition temperature orsoftening temperature, at which point the mold with picoprotuberances isuniformly pressed against the heat-softened material. The mold can beseparated from the substrate after the picoprotuberances are transferredas pico-invaginations into the substrate material. This disclosurepreferably discloses pico-cones and picowells as the patterns of themold and substrate, respectively.

Hot embossing, epoxy masters, and photoresist such as the SU-8photoresist are described (see, Bohl et al (2005) J. Micromechanics andMicroengineering. 15:1125-1130, Jeon et al (2011) Biomed Microdevices.13:325-333, Liu, Song, Zong (2014) J. Micromechanics andMicroengineering. 24:article ID:035009, del Campo and Greiner (2007) J.Micromechanics and Microengineering. 17:R81-R95).

Other microwell plate embodiments. Plastic microwell arrays can bemanufactured by way of a thermal forming using a silicon mold, where thesilicon mold possesses an array of microwells, for example, an array of800,000 microwells. A high degree of control that results in taperedgeometries and smooth sidewalls, and submicron tolerances can be createdwith use of a non-pulsed dry etch process. In contrast, methods that usea pulsed dry etch process, such as the Bosch process, can result inrough sidewalls and lack of control over lateral dimensions duringetching.

Using non-pulsed dry etch process, plastic arrays are fabricated bythermally forming plastic on a silicon master that is created by anon-pulsed isotropic dry etch process using a chrome mask. This processuses three gases, Ar, SF₆, and C₄F₈. The process is conducted at a RFpower between 1200 to 2000 Watts and a bias of 150 Watts. Fine-tuning ofthe taper of the silicon mold with production of smooth sidewalls can beaccomplished by varying the gas flow between the three gases. What isvaried is the ratio of SF₆ to C₄F₈, where the result of changing theratio is, for example, a tapered wall of the mold (the silicon pillar)that resides at an angle of 18 degrees (very slanted walls), 9 degrees(slightly slanted walls), or 2 degrees (walls almost perpendicular tosubstrate) (see, Perry, Henley, and Ramsey (Oct. 26-30, 2014)Development of Plastic Microwell Arrays for Improved ReplicationFidelity. 18^(th) Int. Conference on Miniaturized Systems for Chemistryand Life Sciences. San Antonio, Tex. (pages 1700-1703).

In embodiments, the present disclosure provides a substrate, an array, agrid, a microfluidic device, and the like, that includes an array ofmicrowells. In one embodiment, all of the microwells have essentiallythe same volume. This volume can be about 1 femtoliters, about 2, about4, about 6, about 8, about 10, about 20, about 40, about 60, about 80,about 100, about 200, about 400, about 600 about 800, or about 1,000femtoliters.

Moreover, the volume can take the form of a range between any of theabove two adjacent values, such as, the range of about 40 femtoliters toabout 60 femtoliters. Also, the volume can take the form of a rangebetween any of the above two values that are not immediately adjacent toeach other in the above list.

Furthermore, the volume can be about 1 picoliters, about 2, about 4,about 6, about 8, about 10, about 20, about 40, about 60, about 80,about 100, about 200, about 400, about 600 about 800, or about 1,000,about 2,000, about 5,000, about 10,000, about 20,000, about 50,000,about 100,000, about 200,000, about 500,000, or about 1,000,000picoliters. Also, the volume can take the form of a range between any ofthe above two values that are not immediately adjacent to each other inthe above list.

In exclusionary embodiments, the present disclosure can exclude anysubstrate comprising microwells, or any array comprising microwells,where the volume of each microwell is definable by one of the abovevalues, or is definable by a range of any of the above two values thatare adjacent to each other, or is definable by a range of any of theabove two values that are not adjacent to each other in the list.

Spherical plug (also known as capping beads) on picowells. The presentdisclosure provides a spherical plug, or alternatively, a porousspherical plug, for each and every well, or substantially every well ofa picowell array. A goal of the plug is to keep drugs, drug candidates,cellular contents, and metabolites, inside of the well. The plug alsohelps isolate the contents of picowells from each other. The sphericalplug may need not be perfectly spherical, as long as the goal ofcovering the top (or opening, or mouth), of the picowell may besatisfied. The well can have a top diameter and a bottom diameter.Diameter of spherical plug, prior to capping a well, is about 10micrometers, about 30, about 35, about 40, about 45, about 50, about 55,about 70, about 90, about 120 or about 200 micrometers. The plugs may beadded to cover the picowells by simply flowing them over the picowellarray. Centrifugation, pressure, agitation or other methods may be usedto jam the beads to the tops (or mouths or openings) of the picowells toensure tight sealing. In some embodiments, solvents may be used tomodify the swelling and/or size of the capping beads. In someembodiments, the capping beads may be loaded in a solvent that rendersthe beads shrunken, and once replaced by assay buffer, or a differentsolvent, the capping beads are restored to their original sizes, orswell, thereby sealing the picowells tightly. In some embodimentstemperature may be used to swell or shrink the capping beads to obtainbetter seals at the mouths of picowells. Where needed, capping beads maybe held in place, and prevented from falling further into the picowell,by one of the steps in a stepped picowell array.

The capping beads may be the same type of beads that carry the compoundsof this disclosure, or may be beads of a different type. In someembodiments, the capping beads may actually be the compound bearingbeads themselves. The capping beads may serve as passive caps,preventing or slowing diffusion of molecules out of the picowells, orthe beads may be active beads, where functional moieties attached to thecapping beads may be used to capture reagents from the picowells. Insome embodiments, porous capping beads may passively trap metabolitesreleased from cell-based assays performed inside picowells. In someembodiments, capping beads may non-specifically capture cellularmaterials such as lipid, proteins, carbohydrates and nucleic acids. Insome embodiments, the capping beads may be functionalized withantibodies to specifically capture proteins released from healthy,diseased, lysed or fixed cells. In some embodiments, the capping beadsmay be functionalized with DNA or RNA oligonucleotides that specificallycapture cellular nucleic acids. In some embodiments, the DNA or RNAfunctionalized capping beads may be used to capture microRNA releasedfrom cells within the capped picowells. In some embodiments picowellscontain two beads, a compound containing bead inside the picowell, and acapping bead covering the mouth of the picowells. In some embodiments,the capping beads are also the compound-bearing beads. In someembodiments, the capping beads capture materials released from thecompound beads. In some embodiments, the capping beads capture asampling of the compounds released from compound-beads. In someembodiments, the capping beads capture DNA barcodes released from thecompound-beads. In some embodiments, the capping beads capture differenttypes of analytes released from within the picowells they cap.

Relative hardness of cap and of picowell. A preferred equipment is amicrotiter plate, where each microtiter includes, in its bottom surface,many thousands of picowells. Ability of a cap to seat properly or toseal each picowell can be a function of the hardness of the plastic thatmakes up the picowell's aperture and the picowell's inner walls,relative to the hardness of the cap.

Hardness of a plastic can be defined in terms of a “durometer” value.Hardness is defined and tested as a material's resistance toindentation. The hardness of the spherical plug, and the hardness of thewall of the picowell can be defined in terms of its “durometer.” Thehardness can be, for example, about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95, orabout 100. In attributing any of these durometer values to a plasticsubstance or other substance, one must also state which scale is used.For example, the scale can be ASTM D2240 type A scale, which is used forsofter materials, or the ASTM D2240 type D scale, which is used forharder materials (see, Silicon Design Manual, 6^(th) ed., AlbrightTechnologies, Inc., Leominster, Mass.).

Shapes of picowells. In some embodiments, the picowells may becylindrical picowells where the diameter of the cylinder is roughlysimilar at the top and the bottom of the picowell. In some embodiments,the picowells may have a slight taper, with the top of the picowellsslightly larger than the bottom of the picowells. In some embodiments,the picowells may be conical picowells, with angles off normal anywherebetween 1 degree to 30 degrees. In some embodiments, the picowells arestepped picowells, where the picowells have discontinuous steps from thetop diameter to the bottom diameter (as opposed to conical picowellswhose diameter change smoothly from the top to the bottom). In someembodiments, the stepped picowells have a broad cylinder near theopening of the picowell and a narrower cylinder near the bottom of thepicowells. In some embodiments, the stepped picowells may have multiplediscontinuous steps from the top to the bottom. In some embodiments ofmulti-stepped picowells, the diameter at every rung may be larger thanthe diameter of the rung below it. In some embodiments a small bead maybe deposited at the bottom of the stepped picowell, and a capping beadmay be deposited at the topmost opening of the stepped-picowell. In someembodiments picowells may contain more than 2 beads.

Methods to make stepped-picowells. FIG. 29 disclosed stepped picowell.The embodiment shown has three compartments and two steps. Topcompartment is widest and is configured for accepting cap where most ofthe top compartment is occupied by the cap in the situation where thepicowell is capped. Middle compartment is configured for being occupiedmainly by, or solely by, reagents. Reagents can include buffer, enzymesubstrates, one or more salts, and a preservative or stabilizer such asdithiothreitol, RNAse inhibitor, glycerol, or DMSO. Lowest compartmentis configured for being occupied by bead, that is, a bead with coupledboth a DNA library and with releasable compounds. In addition to bearingDNA barcode and releasable compounds, the same bead can also bear a“response capture element.” Capping beads may be held in place, andprevented from falling further into the picowell, by one of the steps ina stepped picowell. In FIG. 29 , structure 1 is cap., structure 2 isbead, and structure 3 is top region, which is situated immediately abovefirst step. Structure 4 is middle region, which can be used for placingassay reagents. Middle region is immediately above second step. Assayreagents in middle region can diffuse into lowest region. Structure 5 islowest region, which can be used for placing a bead and for placing oneor more cells.

Regarding the space of the lowest compartment that is taken up by thebead (assuming that only one bead is present in picowell), the diameterof the bead can be about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or about 98%, of thediameter of the lowest compartment (assuming that the picowell is acircular well). If picowell is not a circular well, the above values canrefer to widest dimension of the well. In exclusionary embodiments, thepresent disclosure can exclude any system or bead that does not meet anyof the above parameters.

Further regarding space taken up by the bead (assuming that only onebead is present in picowell), about 50% of bead is in lowest compartmentand about 50% of same bead is in middle compartment, where theseparameters can also be: about 55% lowest and about 65% middle, about 60%lowest and about 40% middle, about 65% lowest and about 45% middle,about 70% lowest and about 30% middle, about 75% lowest and about 25%middle, about 80% lowest and about 20% middle, about 85% lowest andabout 15% middle, about 90% lowest and about 10% middle, about 95%lowest and about 5% middle, and about 100% lowest. For making thesecalculations the space taken up by bead assumes (hypothetically) thatthe bead is not porous. In exclusionary embodiments, the presentdisclosure can exclude any system or bead that does not meet any of theabove parameters.

As with conical and cylindrical picowell, using a molding system is onepreferred embodiment to create stepped picowells. For this purpose, amold containing arrays of multilayered pillars is desired, whereuponstamping into a thermoplastic or other curable polymer substrate, animpression of stepped picowells may be formed. A layered pillar arraywith multiple steps, each step of a different diameter (smaller as itgoes up) may be formed by a multilayer lithography process. Briefly, afirst layer of photoresist is exposed, via a first mask, to crosslinkthe first layer of the micropillar array. A second layer of photoresistmay be deposited directly on the (previously exposed) first layer, and asecond photomask may be used to crosslink a second pattern in the secondphotoresist later, and so on. At the end of the multiple-layerpatterning, the stack of resist may be developed to wash away theuncrosslinked regions, leaving an array of multilayered pillars.Detailed protocols for creating multilayered pillar arrays may be foundin Francisco Perdigones et al., (Jan. 8, 2011). Microsystem Technologiesfor Biomedical Applications, Biomedical Engineering, Trends inElectronics Anthony N. Laskovski, IntechOpen. Once an array ofmultilayered pillars array is created, standard processes may be used toimprint stepped picowell arrays using the mold.

Removing the capping beads. In many embodiments it is advantageous tosample the capping beads to study reactions, analytes or cellularresponse to the chemical perturbations within picowells. In someembodiments, the capping beads may be dislodged from the mouths of thepicowells by inverting the picowell array and using mechanicalagitation. In some embodiments solvents may be used to shrink thepicowells, rendering them easier to dislodge from the mouths ofpicowells. In some embodiments, liquids of higher density than thecapping beads may be added on top of the picowell array, causing thecapping beads to raise by buoyancy and float atop the high-densitymedium.

In some embodiments, the capping beads may be crosslinked to each other,converting the capping beads to a capping sheet that can be peeled offthe top of the picowell array. In some embodiments, a crosslinking gelmay be poured over the capped picowells, where the crosslinking gelcrosslinks to the capping beads, and to themselves, causing the cappingbeads to be embedded into a crosslinked sheet that can be peeled off.

Preserving relative locations of picowells, in the form of thepeeled-off layer. It should be appreciated that in such embodiments aswhen the capping beads are enmeshed into a gel layer that can be peeledoff, the relative locations of capping beads with respect to each otherand with respect to the picowells are preserved in the peeled-off layer.This allows direct connection between picowells, assays in picowells,the beads in the picowells, and any materials captured in the cappingbeads.

In some embodiments, fiduial markers may be used to orient the relativefeatures of the picowell arrays to the capping beads in thepeeled-off-layer.

Fiducial markers to enable registration and alignment of picowells.Arranging picowells in irregular arrays allows easy identification ofshifts and drifts during imaging of the picowell arrays. In someembodiments, the picowells are arranged in an irregular order tofacilitate detection of optical and mechanical drifts during imaging. Insome embodiments, the picowells arrays contain fiducial markers to helpidentify shifts and drifts during imaging. In some embodiments, thefiducial markers are easily identifiable shapes, patterns or featuresthat are interspersed between the picowells of the picowell array. Insome embodiments, a small number of picowells may themselves be arrangedin an easily identifiable pattern to allow easy registration in case ofoptical or mechanical drifts during imaging. In some embodiments,external marker, such as fluorescent beads, may be drizzled on thepicowell array to provide fiducial patterns.

Cap-free mat embodiments. Cap-free mat embodiment, at least in someforms or examples, can take the form of a “capless film.” Instead ofsealing openings at the top of picowells, for example, for preventingevaporation of any cell culture medium or enzyme assay medium that maybe in the picowell, sealing can be accomplished by way of a mat.Preferably, the mat is sized to cover all of the picowells in a givenpicowell array. Alternatively, the mat can be sized to cover apredetermined fraction of the picowells in the array. The mat can besecured to the top of the picowell plate, covering picowells and alsocovering the generally planar top surface of the picowell plate thatresides in between the picowells. Secure contact can be achieve by oneor more of: (i) Maintaining constant pressure, for example, by a hardrubber platen that sits on top of and serves as a weight on top of thematt, (ii) Using a mat that is connected to a weight, such as hardrubber platen, (iii) A reversible chemical adhesive, that can be appliedto the entire mat (in the situation where the mat is not be an absorbentmat). Where the mat is to be an absorbent mat, the mat contains circularabsorbent pads that are surrounded by the reversible chemical adhesive.Here, the mat is contacted with the picowell array and aligned so thatthe circular absorbent pads cover only the openings of each picowell,and do not “spill out” over the opening to contact the planar surface ofthe picowell plate.

Membranes for use as mat for contacting substantially planar surface ofpicowell plate, and for use in capless-sealing of picowells, areavailable. Flat sheet membranes, such as Dow Film Tex, GE Osmonics,Microdyn Nadir, Toray, TriSep, Synder, Novamem, Evonik, and Aquaporinflat sheet membranes are available from Sterlitech Corp, Kent, Wash.These include membranes made of polyamide-TFC, cellulose acetate,polyamide-urea-TFC, cellulose acetate blend, polypiperazine-amide-TFC,PES, composite polyamide-TFC, PES, PAN, PVDF, PSUH, RC, PESH, polyetherether ketone, polyimide, and so on. Pore size in terms of molecularweight cutoffs include, 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600 Da,1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da, 20,000 Da,30,000 Da 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da,500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometers, 0.030 micrometers,0.05 micrometers, 0.10 micrometers, 0.20 micrometers, and so on.Regarding the system, compositions, reagents, and methods of the presentdisclosure, these cutoff values can allow selective collection ofcertain classes of compounds with exclusion of other classes ofcompounds. For example, some of the above membranes can allow smallmolecule metabolites to pass through and be absorbed by an absorbablemat, while excluding proteins and other macromolecules. Flat sheetmembranes that are impermeable to all molecules, including water, metalions, salts, metabolites, proteins, and nucleic acids, are alsoavailable for use in the systems, compositions, and methods of thepresent disclosure.

Reversible adhesion can be mediated by “molecular velcro,” for example,metalloporphyrin containing polymers with pyridine-containing polymers(Sievers, Namyslo, Lederle, Huber (2018) eXPRESS Polymer Letters.12:556-568). Other molecular velcro adhesives involve,L-3,4-dihydroxyphenyl alanine, complementary strands of ssDNA (one typeof ssDNA covalently attached to flat upper surface of picowell plate,and other type of ssDNA covalently attached to mat), copolymerscontaining catechol side chains, and so on (see, Sievers, et al, supra).Also, reversible adhesion can be mediated by a gallium adhesive, wheredegree of adhesion can be controlled by slight changes in temperature(Metin Sitti (May 18, 2016) Switch and Stick. The chemical elementgallium could be used as a new reversible adhesive that allows itsadhesive effect to be switched on and off with ease.Max-planck-Gesellschaft). Yet another reversible adhesive is availablefrom DSM-Niaga Technology, Zwoll, The Netherlands.

Absorbent substances (non-specific absorbents, specific absorbents).Absorbent substances, which can be incorporated into a mat to provideabsorbent characteristics include “molecule sieve” beads, such asSepharose®, Sephadex®, Agarose®, as well as ion exchange beads made ofDEAE cellulose, carboxymethylcellulose, phosphocellulose, or anycombination of the above, all combined into one absorbent mat. Absorbentligands include those that are used in high pressure liquidchromatography (HPLC) (see, BioRad catalog, Hercules, Calif.). Specificabsorbents include response-capture elements, such as poly(dT), whichcan capture mRNA by way of hybridizing with polyA tail. Also, responsecapture elements include exon-targeting RNA probes, antibodies, andaptamers. Each or any combination of these can be covalently attached tomat, to create an absorbent mat, where contacting absorbent mat to topsurface of picowell enables capture of aqueous assay medium or aqueouscell culture medium that might be inside picowells.

(IX) Depositing Beads into Picowells

Plates with picowells can take the form of a 96-well plate where each ofthese 96 wells contains many thousands of picowells. Also, plates withpicowells can take the form of a 24-well plate, where each of these 24wells contains many thousands of picowells. For the 96-well plate, eachwell can be filled using 0.1-0.2 mL of a suspension of beads in water orin an aqueous solution. For the 24-well plate, each well can be filledusing about 0.5 mL of a suspension of beads in water or in an aqueoussolution. Suspension can be added using an ordinary pipet with adisposable tip. The number of beads that are in the suspension can bethat resulting in about one third of the picowells containing only onebead, about one third of the picowells containing two beads, and aboutone third of the beads containing either no beads or more than twobeads. Also, the number of beads in the suspension can be that resultingin the situation where, of the wells that do contain one or more beads,at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% of these wells contain only one bead.

After the beads have settled, any excess liquid can be removed bytouching a pipet tip to the wall of each well of the 96 well plate, orby touching a pipet tip to the wall of each well of the 24 well plate,and drawing off the excess liquid.

Regarding assay reagents, where the picowells are to be used forcarrying out reactions, for example, DNA sequencing, biochemical assays,or assays of cultured cells, assay reagents can be added to thepicowells that already contain settled beads. Adding the assay reagentsis with a pipet, as described above for initial addition of the beadsuspensions. After the assay reagents have equilibrated with thesolution that is already in each picowell, any excess solution that isin each of the 96 wells of the 96-well plate, or any excess solutionthat is in each of the 24 wells of the 24-well plate, can drawn off witha pipet tip that touches the wall of each of the 96 wells of the 96-wellplate, or that touches the wall of each of the 24 wells of the 24-wellplate.

Flow-cell embodiment of picowell array. Picowell array may be part of aflow-cell, where a fluidic chamber with an inlet and an outlet aremounted on top of the picowell array. In such embodiments, beads of thisdisclosure, cells, and other assay materials may be flowed in from theinlet and out through the outlet. Gravity or centrifugal force may beused to lodge the beads into the picowells as they are flowed throughthe flowcell.

(X) Sequencing Bead-Bound Nucleic Acids in Picowells

Bead-bound nucleic acids can be sequenced while still attached to beads.Alternatively, or in addition, bead-bound nucleic acids can be sequencedfollowing cleavage of the DNA barcode from the bead.

Cleaving the DNA barcode from the bead before sequencing. In someembodiments, the present disclosure can encompass a method wherebead-bound DNA barcode is cleaved from the bead, thereby releasing theDNA barcode in a soluble form, prior to amplification, or prior tosequencing, or prior to any type of sequence identification techniquesuch as hybridizing with a nucleic acid probe.

Exclusionary embodiments. In embodiments, the present disclosure canexclude any method, associated reagents, system, compositions, or beads,where a bead-bound DNA barcode is cleaved prior to amplification, orprior to sequencing, or prior to any type of sequence identificationtechnique such as hybridizing with a nucleic acid probe. Also, thepresent disclosure can exclude any method where a polynucleotidecomprising a DNA barcode is cleaved, or where a nucleic acid comprisingonly part of a DNA barcode is cleaved, prior to amplification, prior tosequencing, or prior to any type of sequence identification techniquesuch as hybridizing with a nucleic acid probe.

Polymerase chain reaction (PCR), Quantitative PCR (qPCR). The PCRmethod, as well as the qPCR method, depend on the 3-step methodinvolving: (1) Denaturing the DNA template at a high temperature,annealing primers at a reduced temperature, and finally extending theprimer by way of DNA synthesis, as catalyzed by DNA polymerase (Gadkarand Filion (2014) Curr. Issues Mol. Biol. 16:1-6). qPCR is also called,“real time PCR” (Kralik and Ricchi (2017) Frontiers Microbiology. 8 (9pages).

Recent modifications or improvements in the PCR method and qPCR methodinclude, using helicase-dependent (HDA) amplification, using an internalamplification control, using locked nucleic acids (LNA), and usingadditives that bind to inhibitors (Gadkar and Filion (2014) Curr. IssuesMol. Biol. 16:1-6). Locked nucleic acids provide the advantage ofrecognizing and binding its target with extreme precision.

qPCR allows the simultaneous amplification and quantification of atargeted DNA molecule. The qPCR method compares the number ofamplification cycles required for the response curves to reach aparticular fluorescence threshold (Pabinger, Rodiger, Kriegner (2014)Biomolecular Detection Quantification. 1:23-33). Refsland et al providea concise account of apparently typical conditions for conducting qPCR(Refsland, Stenglein, Harris (2010) Nucleic Acids Res. 38:4274-4284).

Guidance is available for designing and validating PCR primers, and onvariables such annealing temperature (Ta), melting temperature (Tm),temperature of elongation step, type of buffer (Bustin and Huggett(2017) Biomolecular Detection Quantification. 14:19-28).

Rolling circle amplification (RCA). DNA can be amplified while attachedto a bead. DNA in amplified form is easier to sequence thatnon-amplified DNA. In the rolling circle amplification method, DNA tags(the DNA barcode) is made single stranded. Once single stranded, asplint oligo is added to bridge the ends of the tag DNA, and this isfollowed by extension and ligation of the splint oligo. Using DNApolymerase (minus 5′→3′exonuclease activity) ensures a ligatablejunction after the DNA catalyzes extension of the splint oligo. Thecircularized DNA can then be subjected to rolling circle amplificationby adding a strand-displacing DNA polymerase, such as phi29 DNApolymerase. The ability to perform rolling circle amplification (RCA) onthe DNA barcode tag permits the use of synthesis chemistries that may bedamaging to DNA, as any surviving DNA molecules can be thermallyamplified to sufficient quantities to be easily sequenced. DNA can bemade single-stranded by exonuclease digestion, nicking, and melting athigh temperature, or by treating with sodium hydroxide.

Further details of rolling circle amplification (RCA) are revealed bythe following steps that can be used for conducting RCA.

Step One: Start with bead-bound ssDNA. If the bead-bound DNA isinitially in a double stranded from (dsDNA), the strand that is not tobe used for RCA can be prepared so that a residue of thymine (T) isreplaced, at or very close to the bead-attachment terminus, with aresidue of uracil (U). If the dsDNA is prepared in this way, uracil-Nglycosidase can be used to cleave the uracil residue, thereby leaving anunstable sugar phosphate (as part of the DNA backbone), where thisunstable location can be cleaved by nuclease-treatment (Ostrander et al(1992) Proc. Natl. Acad. Sci. 89:3419-3423).

Step Two: Add a “splint oligo” to the bead-bound ssDNA. The splint oligois designed so that it hybridizes to about 10-20 base pairs at the end(the 5′-end) of the ssDNA that is covalently coupled to the bead, and sothat it also hybridizes to about 10-20 base pairs at the free end (the3′-end) of the bead-bound ssDNA. The splint oligo does not need to bringthe bead-bound end of the ssDNA in close proximity to the free end ofthe bead-bound ssDNA. All that is needed is for the far ends of thebead-bound ssDNA sequence be tethered together, in order to form a hugeloop.

Step Three: Add sulfolobus DNA polymerase IV, so that this polymeraseuses the huge loop of ssDNA as a template, for creating a complementaryhuge loop that is covalently attached at one end to the splint oligo.

Step Four: Use DNA ligase to covalently close the complementary hugeloop, where the result is circular ssDNA. It is this closed circle ofssDNA that does the “rolling,” during RCA.

Step Five: Add DNA polymerase that has a strand displacement activity,and add dNTPs. The added DNA polymerase covalently attaches dNTPs to thebead-bound ssDNA, and the distal terminus of the bead-bound ssDNA isextended to create a complementary copy of what is on the “rollingcircle,” and then further extended to create yet another complementarycopy of what is on the “rolling circle,” and even more extended tocreate still another complementary copy of what is on the “rollingcircle.” During this process of potentially infinite amplification,continued activity of DNA polymerase is made possible by the stranddisplacement activity of the DNA polymerase.

Optionally, the method of the present disclosure includes real-timemonitoring of rolling circle amplification (RCA) by way of fluorescentmolecular beacons (Nilsson, Gullberg, Raap (2002) Nucleic Acids Res.30:e66 (7 pages)). Reagents for RCA are available from Sigma-Aldrich(St. Louis, Mo.), Sygnis TruePrime Technology (TruePrime® RCA kit),Heidelberg, Germany, and GE Healthcare (TempliPhi 500® amplificationkit). Fluorophores and quenchers are available from ThermoFisherScientific (Carlsbad, Calif.), Molecular Probes (Eugene, Oreg.), CaymanChemical (Ann Arbor, Mich.), and Sigma-Aldrich (St. Louis, Mo.).

Step Six. Use the ssDNA that was amplified by RCA as a template for PCRamplification, where primers are added, where thermostable DNApolymerase is added, and where the PCR products are subsequentlysequenced by Next Generation Sequencing.

In one aspect of the present disclosure, the RCA-amplified ssDNA iscleaved from the bead prior to PCR amplification that makes PCRproducts. In another aspect of the present disclosure, the PCRamplification that makes PCR products can be made without cleaving theRCA-amplified ssDNA from the bead.

As described by Baner et al, “Through the RCA reaction, a strand can begenerated that represents many tandem copies of the complement to thecircularized molecule” (Baner, Nilsson, Landegren (1998) Nucleic AcidsRes. 26:5073-5078). Bacillus subtilis phase phi29 DNA polymerase is asuitable enzyme, because of its strand displacement activity and highprocessivity. RCA is similarly characterized by Li et al as, “In RCA, acircular template is amplified isothermally by a DNA polymerase phi29with . . . strand displacement properties. The long single-stranded DNAproducts contain thousands of sequence repeats: (Li and Zhong (2007)Anal. Chem. 79:9030-9038).

Sequencing of DNA barcodes of the present disclosure can be, withoutimplying any limitation, with methods of Vander Horn U.S. Pat. No.8,632,975, which is incorporated herein by reference in its entirety.Also, the DNA barcodes of the present disclosure can be sequenced, forexample, by methods that use sequencing-by-synthesis, such as the Sangersequencing method, or by methods that use “Next Generation sequencing.”

Illumina method for DNA sequencing. Illumina method for DNA sequencingis as follows. DNA can be fragmented to a size range of 100-400 basepairs (bp) by sonication (Hughes, Magrini, Demeter (2014) PLoS Genet.10:e1004462). In the Illumina method, DNA libraries are made, wherefragments of DNA from a cell or from cells are modified by DNA adaptors(attached to termini of the fragments). The reaction product takes theform of a sandwich, where the DNA to be sequenced is in the center ofthe sandwich. The reaction product takes the form: (first adaptor)-(DNAto be sequenced)-(second adaptor). The adaptor-DNA-adaptor complex isthen associated with yet another adaptor, where this other adaptor iscovalently attached to a solid surface. The solid surface can be a flatplate. The solid surface has a lawn of many adaptors that stick out ofthe flat surface. The adaptor has a DNA sequence that is complementaryto one of the adaptors that is in the sandwich. Actually, the lawncontains two type of adaptors, where one adaptor binds (hybridizes) toone of the adaptors in the complex, and non-covalently tethers thecomplex to the plate. These may be called the, “first lawn-boundadaptor” and the “second lawn-bound adaptor.” The first task of DNApolymerase, is to create a daughter strand, using the tethered (butnon-covalently bound) DNA as a template and, when DNA polymerizationoccurs, the daughter strand is in a form that is covalently attached tothe “first lawn-bound adaptor.” This covalent link was generated by thecatalytic action of DNA polymerase. After the daughter strand iscompletely synthesized, the distal end (the end that sticks out into themedium) contains a DNA sequence that is complementary to the secondadaptor in the above-named sandwich. This DNA sequence that iscomplementary, allows the distal end of the newly synthesized daughterDNA to bend over and to hybridize to the “second lawn-bound adaptor.”What has been described above, is how both adaptors of the sandwich areused, and how both the “first lawn-bound adaptor” and the “secondlawn-bound adaptor” are used.

A cycle of reactions is then performed many times, where the result is acluster of amplified versions of the original dsDNA. Actually, thecluster takes the form of covalently attached (tethered) ssDNAmolecules, where all of these ssDNA molecules correspond to only one ofthe strands of the original dsDNA (dsDNA isolated from a living cell ortissue). This cluster of tethered ssDNA molecules is called a “polony.”The generation of the polony is by a technique called, “bridgeamplification.” Finally, after bridge amplification and the creation ofpolonies, the reverse strands that are covalently attached to the solidsurface are cleaved from its tetherings, washed away, and discarded,leaving only the forward strands.

Information on the Illumina® method is available from Goodwin,McPherson, McCombie (2016) Nature Rev. Genetics. 17:333-351, Gierahn,Wadsworth, Hughes (2017) Nature Methods. 14:395-398, Shendure and Hanlee(2008) Nature Biotechnology. 26:1135-1145, Reuter, Spacek, Snyder (2015)Molecular Cell. 58:586-597, Illumina Sequencing by Synthesis (5 minutevideo on YouTube).

Sequencing by oligonucleotide ligation and detection (SOLiD sequencing).SOLiD measures fluorescence intensities from dye-labeled molecules todetermine the sequence of DNA fragments. A library of DNA fragments isprepared from the sample to be sequenced and used to prepare clonal beadpopulations (with only one species of fragment on the surface of eachmagnetic bead). The fragments attached to the beads are given auniversal P1 adapter sequence attached so that the starting sequence ofevery fragment is both known and identical. PCR is conducted and theresulting PCR products that are attached to the beads are thencovalently bound to a slide.

Then, primers hybridize to the P1 adapter sequence within the librarytemplate. A set of four fluorescently labelled di-base probes competefor ligation to the sequencing primer. Specificity of the di-base probeis achieved by interrogating every 1st and 2nd base in each ligationreaction. Multiple cycles of ligation, detection and cleavage areperformed with the number of cycles determining the eventual readlength. Following a series of ligation cycles, the extension product isremoved and the template is reset with a primer complementary to the n−1position for a second round of ligation cycles (see, Wu et al (2010)Nature Methods. 7:336-337).

pH-based DNA sequencing. pH-Based DNA sequencing is a system and methodwhere, base incorporations are determined by measuring hydrogen ionsthat are generated as byproducts of polymerase-catalyzed extensionreactions. DNA templates each having a primer and polymerase operablybound are loaded into reaction chambers or microwells, after whichrepeated cycles of deoxynucleoside triphosphate (dNTP) addition andwashing are carried out. The DNA template is templates are attached asclonal populations to a solid support. With each such incorporation ahydrogen ion is released, and collectively a population of templatesreleasing hydrogen ions causing detectable changes to the local pH ofthe reaction chamber (see, Pourmand (2006) Proc. Nat'l. Acad. Sci.103:6466-6470). The present disclosure can exclude pH-based DNAsequencing.

Regarding the concatenated DNA barcode, the entire concatenated DNAbarcode can be sequenced in one run (where sequencing of the entireconcatenated DNA barcode requires only one sequencing primer).Alternatively, some or all of the DNA barcode modules that make up theconcatenated DNA barcode can be subjected to individual sequencing(where each of the individually-sequenced DNA barcode modules gets itsown sequencing primer). Regarding orthogonal DNA barcodes, each of theDNA barcode modules that make up the orthogonal DNA barcode needs itsown, dedicated sequencing primer, because of the fact that each DNAbarcode module is attached to its own site on the bead.

Exclusionary embodiments. In embodiments, the present disclosure canexclude any system, device, combination of devices, and method, thatinvolves microfluidics, aqueous droplets that reside in an oil medium,and aqueous droplets that are created where a first channel containingaqueous reagents is joined with a second channel containing an oil tocreate aqueous droplets that move through an oil medium through a thirdchannel that starts at the joining area. Microfluidics devices andreagents are described (see, e.g., Brouzes, Medkova, Savenelli (2009)Proc. Natl. Acad. Sci. 106:14195-14200, Guo, Rotem, Hayman (2012) LabChip. 12:2146-2155, Debs, Utharala, Balyasnikova (2012) Proc. Natl.Acad. Sci. 109:11570-11575, Sciambi and Abate (2015) Lab Chip.15:47-51).

In other exclusionary embodiments, what can be excluded is any reagent,composition, nucleic acid, or bead, that comprises a “DNA headpiece” oran reagent, composition, nucleic acid, or bead, that is covalentlyattached to a “DNA headpiece.” MacConnell, Price, Paegel (2017) ACSCombinatorial Science. 19:181-192, provide an example of a DNAheadpiece, where beads are functionalized with azido DNA headpiecemoieties.

Additional exclusionary embodiments relating to sequencing methods andsequencing reagents. In embodiments, the present disclosure can excludereagents, systems, or methods that do not involve use of a “reversibleterminator” in DNA sequencing. Also, what can be excluded is anyreagent, system, or method, that do not include methoxy blocking group.Moreover, what can be excluded is any reagent, system, or method, thatinvolves DNA sequencing, but where the DNA being sequenced is notcovalently bound to a bead at the time at the time that information onthe order of polynucleotides is being detected and collected.Furthermore, what can be excluded is any reagent, system, or method thatamplifies a DNA template prior to conducting sequencing reactions, forexample, amplification by PCR technique or by rolling circle technique.In embodiments, what can be excluded is any method of barcoding, forexample, nucleic acid barcoding, that is concatenated (all informationon synthesis of a member of the chemical library residing on one singlenucleic acid). In another aspect, what can be excluded is any method ofbarcoding, for example, nucleic acid barcoding, that is orthogonal(information on synthesis of a given monomer of a chemical library beingdispersed on a plurality of attachment positions on the bead). In anexclusionary embodiment relating to DNA ligase, the present disclosurecan exclude any reagent, system, or method, that uses DNA ligase forconnecting modules of a nucleic acid barcode.

Fluorophores, quenchers, and FRET-based assays. The present disclosureprovides fluorophores and quenchers for screening members of a chemicallibrary, or for characterizing an isolated member of a chemical library.FRET is Förster resonance energy transfer.

Assays can be performed on bead-bound chemical libraries. Also, assayscan be performed on free chemical library members shortly after cleavagefrom a bead, that is, performed in the same microwell as the bead orperformed in the same vicinity of a hydrogel matrix as the bead.Moreover, assays can be performed on a soluble chemical library memberthat had never been attached to any bead, or that had been cleaved froma bead and then purified.

Fluorophores suitable as reagents of the present disclosure includeAlexa 350, Alexa 568, Alexa 594, Alexa 633, A647, Alexa 680,fluorescein, Pacific Blue, coumarin, Alexa 430, Alexa 488, Alexa 532,Alexa 546, Alexa 660, ATTO655, ATTO647n, Setau-665 (SETA Biochemicals,Urbana, Ill.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, tetramethylrhodamine (TMR),Texas red, tetrachlorofluorescein (TET), hexachlorofluorescein (HEX),and Joe dye (4′-5′-dichloro-2′,7′-dimethoxy-6-carboxyfluorescein), SYBRgreen I (absorb 497 nm, emit 520 nm), 6-carboxyfluorescein (6-FAM)(absorbs 492 nm, emits 518 nm), 5-carboxyfluorescein (5-FAM) (absorbs492 nm, emits 518 nm), FITC, and rhodamine. Quenchers include TAMRAquencher, black hole quencher-1 (BHQ1), and black hole quencher-2(BHQ2), and DABCYL quencher. Please note, as disclosed elsewhere in thispatent document, that TAMRA can be a fluorophore and it can also be aquencher.

Guidance is available on reagents for FRET-based assays, where the FRETreagent includes a fluorophore and quencher (see, Johansson (2006)Choosing reporter-quencher pairs for efficient quenching. Methods Mol.Biol. 335:17-29). An example of a FRET-based assays including measuringthe activity of a signal peptidase (SpsB) with the substrate, “SceDpeptide.” The FRET-pair attached to the peptide was4-(4-dimethylaminophenylazo) 5-((2-aminoethyl)amino)-1-nepthalenesulfonic acid (see, Rao et al (2009) FEBS J.276:3222-3234). Another example comes from assays of HIV-1 protease,with the peptide substrate, KVSLNFPIL. The donor/acceptor FRET pair wasEDANS (donor) and DABCYL (acceptor). EDANS fluorescence can be quenchedby DABCYL by way of resonance energy transfer to the nonfluorescentDABCYL (see, Meng et al (2015) J. Biomolecular Screening. 20:606-615).Yet another example comes from assays of botulinum toxin. Activity ofSNAP-25 can be measured by using the substrate, BoNT-A. For FRET-basedassays, the substrate had an N-terminally linkedfluorescein-isothiocyanate (FITC) and the C-terminally linked quencherwas, 4-(4-dimethylaminophenyl) diazenylbenzoic acid (DABSYL). Thepeptide substrate corresponded to amino acids 190-201 of SNAP-25 (see,Rasooly and Do (2008) Appl. Environ. Microbiol. 74:4309-4313).

The present disclosure provides for reagents, compositions, and methodsfor screening a library of compounds in order to discover and identifyenzyme inhibitors, enzyme activators, and to discover compounds that canenhance the rate of in vivo degradation of a given protein. Thesereagents, compositions, and methods can use FRET-based assays and,alternatively, they can use assays other than FRET-based assays.

Molecular beacons are described (see, Baruch, Jefferey, Bogyo (2004)Trends Cell Biology. 14:29-35). A molecular beacon is a reagent where afluorophore is bound, by way of a linker, to a quencher. The linker maybe cleavable by a nuclease, and thus measure nuclease activity. Thepresent disclosure provides for methods to screen chemical libraries foridentifying nuclease inhibitors and, alternatively, for identifyingnuclease activators. Feng et al have described the use of molecularbeacons and use of FRET-based assays for measuring activity of variousnucleases (Feng, Duan, Liu (2009) Angew Chem. Int. Ed. Engl.48:5316-5321). Feng et al, showed use of FRET-based assays for measuringactivity of various restriction enzymes.

(XI) Releasing Bead-Bound Compounds

Cleavable linkers. What is provided is linkers that are not cleavable.Also, what is provided are cleavable linkers (see, Holmes and Jones((1995) J. Org. Chem. 60:2318-2319, Whitehouse et al (1997) TetrahedronLett. 38:7851-7852, and Yoo and Greenberg ((1995) J. Org. Chem.60:3358-3364, as cited by Gordon et al (1999) J. Chem. TechnologyBiotechnology. 74:835-851). Cleavable linkers also include an acylsulphonamide linkers that reside alkaline hydrolysis, as well asactivated N-alkyl derivatives which are cleaved under mild conditions,and also traceless linkers based on aryl-silicon bonds, and tracelesslinkers based on silyl ether linkages (described on page 839 and 842 ofGordon et al (1999) J. Chemical Technology Biotechnology. 74:835-851).Moreover, what is provided is a linker based on tartaric acid which,upon cleavage, generates a C-terminal aldehyde, where cleavage is byperiodate oxidation (see, Paulick et al (2006) J. Comb. Chem.8:417-426).

FIG. 3 discloses various cleavable linkers that are suitable for thecompositions and methods of the present disclosure. FIG. 3 is reproducedfrom Table 1 of: Yinliang Yang (2014) Design of Cleavable Linkers andApplications in Chemical Proteomics. Technische Universitat MunchenLehrstuhl fur Chemie der Biopolymere. From FIG. 3 , cleavable linkersthat are preferred for the present disclosure are linkers a, c, d, p, q,r, and t. Linker p was used in the experimental results disclosedherein. Cleavage conditions for these are DTT (linker a), Na₂SO₄ (linkerc), Na₂SO₄ (linker d), UV light (linker p), UV light (linker q), UVlight (linker r), and TEV protease (linker t). These particular cleavageconditions are gentle and are not expected to damage the bead, to damagethe bead-bound compound, or to damage any chemical library member (theunit) of the bead-bound compound.

Chemically cleavable linkers that are compatible with click-chemistry.Qian et al (2013) describes a number of cleavable linkers that arecompatible with click-chemistry (Qian, Martell, Pace (2013) ChemBioChem.14:1410-1414). These include linkers with an azo bond, where the azobond is cleavable with dithionite. This linker has the followingstructure: R₁-benzene1-N═N-benzene₂-R₂. The first benzene ring has ahydroxy group para to R₁, and the second benzene ring has a carbonylgroup that links to R₂, where this carbonyl group is para to the azomoiety.

Photolabile cleavable linkers. The present disclosure encompassesphotocleavable linkers that have an o-nitrobenzyl group. This group canbe cleaved by irradiation at 330-370 nm (see, Saran and Burke (2007)Bioconjugate Chem. 18:275-279, Mikkelsen, Grier, Mortensen (2018) ACSCombinatorial Science. DOI:10.1021). A linker with a shorter photolysistime than o-nitrobenzyl linker is 2-(2-nitrophenyl)-propyloxycarbonyl(NPPOC) linker. A variation of o-nitrobenzyl linker iso-nitrobenzylamino linker. When attached to a peptide chain, and whensubsequently cleaved, this linker releases an amide. Linker with ano-nitroveratryl group are available, and these have shorter photolysistime and greater release yields than unsubstituted o-nitrobenzyllinkers. Also available are phenacyl linkers, benzoin linkers, andpivaloyl linkers (see, Mikkelsen et al (2018) ACS Combinatorial Science.DOI:10.1021).

Linkers with photocleavable ether bonds are available. Thisphotocleavable linker can be used where the linker is attached to a beadand where the cleavable group is an “R group,” and after cleavage, thereleased group takes the form of ROH (see, Glatthar and Giese (2000)Organic Letters. 2:2315-2317). Also available are linkers withphotocleavable ester bonds (see, Rich et al (1975) 97:1575, Renil andPillai (1994) Tetrahedron Lett. 35:3809-3812, Holmes (1997) J. Org.Chem. 62:2370-2380, as cited by Glatthar and Giese, supra). Ether bondsin linkers can be cleaved by acid, base, oxidation, reduction, andfluoride sensitive silyl-oxygen bond cleavage, and photolysis (Glattharand Giese, supra).

Another photocleavable linker, which has been used to link a peptide(R₁) and a nucleic acid (R₂), is as follows. R₁ is connected directly tothe methylene moiety of a benzyl group. Para to the methylene group is aring-attached nitro group. Meta to the methylene moiety is aring-attached ethyl group. The 1-carbon of the ethyl group bears aphosphate. To an oxygen atom of this phosphate is attached the R₂ group(Olejnik et al (1999) Nucleic Acids Res. 27:4626-4631).

Akerblom et al, discloses photolabile linkers of the alpha-methyl2-nitrobenzyl type, containing amino, hydroxyl, bromo, and methylaminogroups, and also 4-nitrophenoxycarbonyl activated hydroxyl and aminogroups (see, Akerblom and Nyren (1997) Molecular Diversity. 3:137-148).Cathepsin B can cleave a linker with the target sequence,“valine-citrulline” (Dal Corso, Cazzamalli, Neri (2017) BioconjugateChemistry. 28:1826-1833).

Enzyme-cleavable linkers. Linkers that are cleavable by enzymes, such asproteases, are available (see, Leriche, Chisholm, Wagner (2012)Bioorganic Medicinal Chem. 20:571-582). The hydroxymethylphenoxy linkercan be cleaved with chymotrypsin (Maltman, Bejugam, Flitsch (2005)Organic Biomolecular Chem. 3:2505-2507). Linkers that are cleavable withtobacco etch virus protease are available (see, Weerapana, Speers,Cravatt (2007) Nature Protocols. 2:1414-1425, Dieterich, Link, Graumann(2006) Proc. Nat'l. Acad. Sci. 103:9482-9487). The linker sequencesLVPRG and LVPRGS can be cleaved by thrombin (Jenny, Mann, Lundblad(2003) Protein Expression Purification. 31:1-11). Plasmin-cleavablelinkers are available (Devy, Blacher, Noel (2004) FASEB J. 18:565-567).

Bead-bound release-monitor. The present disclosure provides a novel andunique release-monitor that is capable of assessing release ofbead-bound compounds. The release-monitor takes the form of a bead-boundcomplex of fluorophore and quencher, where the fluorophore is connectedto the bead by way of a cleavable linker. Preferably, the cleavablelinker is a photocleavable linker. Preferably, the bead-boundrelease-monitor is situated in a dedicated picowell, where that picowelldoes not contain any other type of bead. With severing of thephotocleavable linker, the fluorophore is released from the bead,diffuses into the medium in the picowell, achieves some distance fromthe bead-bound quencher, where the result is an increase in fluorescencethat is proportional to the amount of release. The increase influorescence allows the calculation of the concentration of the freefluorophore that is in the picowell and, more importantly, allowscalculation of the amount of chemical compounds that are released fromother beads that are situated in other wells.

To summarize, the bead-bound release-monitor is situated in its owndedicated well, where other wells contained bead-bound compounds thatare drug candidates.

FIG. 8 discloses a simplified version of a preferred and non-limitingexample of a bead-bound release-monitor. The release-monitor takes theform of a quencher that is held in the vicinity of a fluorophore,resulting in quenching of the fluorophore. In embodiments, quenching isat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%,at least 99.95%, and so on. In a picowell, one bead is dedicated tobeing a release-monitor, while another bead or beads are used forattaching a compound and for attaching DNA library. Exposure of all ofthe beads in a picowell to UV light result in simultaneous cleavage offluorophore and of the compound. QSY7 is a preferred quencher. Thestructure and CAS number for QSY7 is as follows (see below):

CAS name/number: Xanthylium,9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-,chloride 304014-12-8

The increase in fluorescence that results from separation of thefluorophore from the quencher can be used to infer the concentration inthe picowell of the simultaneously released compound. Also, the increasein fluorescence that results from separation of the fluorophore from thequencher can be used to infer the number of molecules (molecules takingthe form of the compound that was formerly a bead-bound compound) thatreside in free form in the picowell. In a more preferred embodiment, therelease-monitor comprises a quencher and a fluorophore, where cleavageresults in the release of the fluorophore (and not release of thequencher). This embodiment provides lower background noise than thefollowing less preferred embodiment. In a less preferred embodiment,cleavage results in the release of the quencher, where the read-outtakes the form of the increase in fluorescence from bead-boundfluorophore.

The release-monitor provides the user with a measure of theconcentration of the soluble compound, following UV-induced release ofthe compound from the bead. In a preferred embodiment, one type of beadis dedicated to being a release-monitor. By “dedicated,” what this meansis that this bead does not also contain bead-bound compound and does notalso contain bead-bound DNA library.

As a general proposition, just because a compound has been released froma bead by cleavage of a photosensitive linker, it should not be inferredthat the compound has become a soluble compound. First of all, pleasenote that just because a compound is considered to be “hydrophobic” oris considered to be “water-insoluble” does not mean that none of themolecules are freely moving in the solvent. For example, evencholesterol has a measurable solubility in water (see, Saad and Higuchi(1965) Water Solubility of Cholesterol. J. Pharmaceutical Sciences.54:1205-1206). Moreover, biochemical efficacy of a bead-boundwater-insoluble compound can be increased, by way of surfactants,detergents, additives such as DMSO, or carriers such as human serumalbumin. Thus, the release-monitor can be used to assess overallconcentration of compounds of limited water-solubility or of nowater-solubility, under the condition where the picowell contains one ofthe above agents or, alternatively, where the water-insoluble compoundis released in the vicinity of the plasma membrane of a living cell thatis cultured inside of the picowell.

FIG. 9 discloses a simplified version of a preferred embodiment ofbead-bound release-monitor, while FIG. 10 discloses a complete anddetailed structure of this preferred embodiment of bead-boundrelease-monitor.

FIG. 30 provides data demonstrating use of bead-release monitor, wherebead is in a picowell. The bead-bound fluorophore, which is bound usinga light-cleavable linker, was TAMRA (excitation wavelength 530 nm,emission wavelength 570 nm). The figure shows time-course of release ofthe fluorophore from the bead. This shows operation of the bead-boundrelease monitor, acquisition of fluorescent data at t=0 seconds, t=1seconds, t=11 seconds, and t=71 seconds. FIG. 30 also includes insetsshowing blowups of the smaller figures, for two of the four smallerfigures. FIG. 30 was obtained from incubation of cathepsin-D, which isan aspartyl protease, with “Peptide Q-Fluor Substrate” and beads.Reagents were placed into wells at 4 degrees C. Ultraviolet light at 365nm was used to cleave the fluorophore from the bead, thereby releasingthe fluorophore and separating it from the quencher. A goal of thisassay was to assess the time course of release taking place in aseparate well, where the separate well contained a different type ofbead. The different type of bead had the same light-cleavable linker,but where this light-cleavable linker was attached to pepstatin-A.Release of pepstatin-A can bind to and inhibit an aspartyl protease thatis in the same assay medium. This setup with bead-bound pepstatin-A andthe aspartyl protease can serve as a positive control.

UV exposed through 20× objective. Image was obtained with Gain=5,Exposure was 400 ms. Excite at TAMRA at 530 nm. TAMRA emits at 570 nm.

FIG. 35 discloses further details on enzymatic assays, where bead-boundpepstatin-A is released, and where the released pepstatin-A results inenzyme inhibition. 10 μm TentaGel beads displaying photocleavablePepstatin-A (positive control) and a covalent Cy5 label, were mixed with10 μm TentaGel beads displaying photocleavable Fmoc-Valine (negativecontrol) in PBST buffer. This bead population was introduced intopicowells, then buffer exchanged into a protease inhibition assay,including Cathepsin-D protease and Peptide Q-Fluor substrate (λ_(ex)=480nm, λ_(em)=525 nm). Wells were encapsulated by air, and entire slideexposed to UV (365 nm, 77 J/cm²), cleaving the photolabile linker,releasing the compound to reach approximately 13 μM. The flowcell wasincubated (30 min, 37° C.). Wells containing positive control beadsshould inhibit peptide proteolysis by Cathepsin-D, resulting in lowfluorescence signal. Wells containing negative control beads should notshow any Cathepsin-D inhibition, and should be similar in fluorescenceintensity to empty wells.

Terminology for quencher and fluorophore can change, for a givenchemical, depending on what other chemicals occur in the immediatevicinity. Although the TAMRA that is used in the laboratory data of thebead-bound release monitor is a fluorophore, in other contexts, TAMRAcan be a quencher. TAMRA acts as a quencher in TaqMan® probes thatcontain FAM and TAMRA.

Additional accounts of experimental setup and laboratory data. Thepresent disclosure provides data on controlled5(6)-Carboxytetramethylrhodamine (TAMRA) concentrations in phosphatebuffer (10 mM phosphate, 154 mM sodium, pH 8.0) within filledpico-wells, compartmentalized by air. Fluorescence images captured (10ms, 2 ms exposures) and well-area quantitated by mean pixel intensity(n≥100) to generate a concentration vs fluorescence intensitycalibration curve. The above data take the form of a standard curve,showing fluorescence at various predetermined concentrations of freeTAMRA (2, 10, 30, 60, 100 mM TAMRA). This standard curve was preparedunder two different conditions, that is, where the photographic imagewas taken with a 2 millisecond exposure or with a 10 millisecondexposure. The experiment used for preparing the standard curve wasconducted in picowells, but there were not any beads used in thisexperiment (just known amounts of TAMRA). The photographic image is notshown in this patent document, because the data merely take the form ofa standard curve, which may also be called a calibration curve.

The experimental setup included the following. For Scheme X),TentaGel-Lys(PCL1-Tamra)-QSY7 bead structure. QSY7 (gray) quenches theTamra fluorophore (orange) while covalently attached to bead through aphotocleavable linker (purple). Irradiation from UV (365 nm) providesquantitative release of compounds in situ

FIG. 31 discloses emission data resulting after catalytic action ofaspartyl protease on quencher-fluorophore substrate. Greaterfluorescence means that the enzyme is more catalytically active. Lesserfluorescence means that the enzyme is less catalytically active, thatis, there the enzyme is more inhibited by a free inhibitor, where theinhibitor was freed from a bead, and where freedom was obtained bycleavage of light-cleavable linker. Images were captured following UVrelease and Cathepsin-D assay incubation (λ_(ex)=480 nm, λ_(em)=525 nm).Wells containing positive control beads could be identified spectrallyby Cy5 fluorophore (λ_(ex)=645 nm, λ_(em)=665 nm, orange false color). Asection was analyzed with a line-plot across open well volume, Wellscontaining negative control beads elicit no Cathepsin-D inhibition.Assay volume within wells containing positive control beads are dark,indicating strong inhibition. Assay volume within empty wells iscomparable to wells containing negative control beads.

FIG. 32 illustrates the following procedure. Further regarding SchemeX), Picowell substrate (46 pL per well) is enclosed in a flowcell, wellswetted under vacuum, a suspension of TentaGel-Lys(PCL1-TAMRA)-QSY7 beadsare introduced, and air pulled across flow-cell, compartmentalizing eachwell (top). Flowcell is irradiated by a UV LED (λ_(mean) 365 nm) withcontrolled luminous flux, allowed to equilibrate (20 min), beforefluorescence microscopy images taken to quantitate released compound(TAMRA) concentration (bottom) (FIG. 32 ). In detail, FIG. 32 showsdrawings of cross-section of picowell, illustrating the steps wherepicowells wetted in a flowcell, the step where beads in a suspension areintroduced over the picowells, resulting in one bead per picowell, thestep of drawing air across flowcell in order to reduce excessivedispersion solution and resulting in a meniscus dropping below thesurface of the planar top surface of the picowell plate, the step ofcontrolled UV exposure (365 nm), resulting in release of some TAMRA, andthe step of provoking light emission from TAMRA with detectingfluorescent signal with fluorescent microscopy (excite 531/40 nm) (emit594/40 nm). The notation, “slash 40” refers to the bandwidth, that is,it means that cut-off filters confined the light to the range of: 531 nmplus 20 nm and minus 20 nm, and to 594 nm, plus 20 nm and minus 20 nm(this slash notation can be used for excitation wavelengths and also toemission wavelengths).

The present inventors acquired photographs showing the following data(see, FIG. 33 ). Fluorescence emission (λ_(ex) 531/40 nm, λ_(em) 593/40)of fluorophore (TAMRA) released from 10-μm TentaGel-Lys(PCL1-TAMRA)-QSY7beads after UV LED (365 nm) exposure in pico-well flow cell. A) Nosignificant emission above background prior to UV exposure (0 J/cm²),owed to the FRET quenching effect of QSY7. TAMRA release allowed toreach equilibrium (20 min) following UV exposures of (B) 25 J/cm², (C)257 J/cm², (D) 489 J/cm², (E) 721 J/cm², (F) 953 J/cm² then imaged usingappropriate exposure times. Fluorescence emission was measured withinthe volume surrounding each bead to measure TAMRA concentration (FIG. 33) The notation, “slash 40” refers to the bandwidth, that is, it meansthat cut-off filters confined the light to the range of: 531 nm plus 20nm and minus 20 nm (this slash notation can be used for excitationwavelengths and also to emission wavelengths).

The following is an interpretation, by the present inventors, of some ofthe fluorescence data from testing and use of the bead-bound releasemonitor (see, FIG. 34 ) Concentration of bead-released TAMRA insidepico-wells (45 pL) after UV exposure (365 nm). Image analysis used meanpixel intensity of the solution surrounding bead-filled wells (n≥14),normalized to image exposure time, then correlated to standard curve ofknown TAMRA concentrations in pico wells. Error bars represent 1c,calculated from RSD %. UV released compound concentrations were 1.1 μM(RSD % 8.9), 54.3 μM (RSD % 5.2), 142 μM (RSD % 4.2), 174 μM (RSD %7.7), 197.3 μM (RSD % 10.1) (FIG. 34 )

(XII) Biochemical Assays for Compounds (Assays that are not Cell-Based)

A variety of biochemical assays are possible using beads withinpicowells. Non-limiting examples include binding assays, enzymaticassays, catalytic assays, fluorescence based assays, luminescence basedassays, scattering based assays, and so on. Examples are elaboratedbelow.

Biochemical assays that are sensitive to inhibitors of proteases andpeptidases. Where the goal is to detect and then develop a drug thatinhibits a protease, screening assay can use a mixture of a particularprotease or peptidase, a suitable cleavable substrate, and a color-basedassay or a fluorescence-based assay that is sensitive to the degree ofinhibition by candidate drug compounds. For example, one reagent can bea bead-bound compound, where the compound has not yet been tested foractivity. Another reagent can take the form of bead-bound pepstatin (anestablished inhibitor of HIV-1 protease) (Hilton and Wolkowicz (2010)PLoS ONE. 5:e10940 (7 pages)). Yet another reagent can be a cleavablesubstrate of HIV-1 protease, and where cleavage by the HIV-1 proteaseresults in a change in color or a change in fluorescence.Positive-screening drug candidates are identified where a particularassay (in a given microwell) results in a difference in color (or adifference in fluorescence). The cleavable substrate takes the form of asusceptible peptide that is covalently bound to and flanked by aquencher and a fluorescer. Before cleavage, the fluorophore does notfluoresce, because of the nearby quencher, but after cleavage,fluorescence materializes (see, Lood et al (2017) PLoS ONE. 12:e0173919(11 pages), Ekici et al (2009) Biochemistry. 48:5753-5759, Carmona et al(2006) Nature Protocols. 1:1971-1976). The reagents and methods of thepresent disclosure encompass the above-disclosed technology.

Enzyme-based screening assay for compounds that inhibit ubiquitinligases, where the reagents include MDM2 (enzyme) and p53 (substrate).Applicants have conducted working tests based on the followingtechnology. MDM2 regulates the amount of p53 in the cell. MDM2 isoverexpressed in some cancers. MDM2 is an enzyme, as shown by thestatement that, “In vitro studies have shown that purified MDM2 . . . issufficient to ubiquitinate . . . p53” (Leslie et al (2015) J. Biol.Chem. 290:12941-12950). Applicant's goal is to discover inhibitors ofMDM2, where these inhibitors are expected to reduce ubiquitination ofp53 and thus reduce subsequent degradation of p53. In view of theexpected increase in p53 in the cell, an inhibitor with the aboveproperty is expected to be useful for treating cancer.

Applicants used the following enzyme-based assay for assessing theinfluence of lenalidomide on ubiquitination of p53, as mediated byMDM2/HDM2. Applicants used reagents from the following kit: MDM2/HDM2Ubiquitin Ligase Kit—p53 Substrate (Boston Biochem, Cambridge, Mass.).One of the reagents used in the assay was a bead with a covalently boundantibody. The bead was TentaGel® M NH₂ (cat. no. M30102, Rapp PolymereGmbH, Germany) and the antibody was anti-human p53 monoclonal antibody,biosynthesized in a mouse. MDM2 is an E3 ligase that can use p53 as asubstrate, where MDM2 catalyzes ubiquitination of the p53.

Goal of activating p53 for reducing cancer. A relation between MDM2, thetranscription factor called, “p53,” and anti-cancer therapy is suggestedby the following description. The description is, “MDM2 is an E3ubiquitin ligase that ubiquitinates p53, targeting it for proteasomaldegradation” (Ortiz, Lozano (2018) Oncogene. 37:332-340). p53 hastumor-suppressing activity. p53 activity can be inhibited by MDM2.According to Wu et al, MDM2 is a, “p53-binding protein” (see, Wu,Buckley, Chernov (2015) Cell Death Disease. 6:e 2035). Where a compoundprevents ubiquitination of p53, for example, by blocking interactionsbetween MDM2 and p53, the compound might be expected to function as ananti-cancer drug.

Goal of the screening assay. A purpose of the screening assay is todiscover compounds that influence ubiquitination of p53, for example,compounds that stimulate p53 ubiquitination and compounds that inhibitp53 ubiquitination. In detail, the purpose is to discover compounds thatare inhibiting or activating, where their effect is via MDM-2 and eitherE1 ligase, E2 ligase, or E3 ligase. MDM2 means, “murine double minute.”MDM2 has been called an, “E3 ubiquitin ligase.” When MDM2 occurs in thecell, evidence suggests its activity in catalyzing the ubiquitination ofp53 requires a number of other proteins, such as CUL4A, DDB1, and RoC1(see, Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729, Nag et al (2004)Cancer Res. 64:8152-8155). Banks et al have described a physicalinteraction involving p53 and MDM2 as, “L2DTL, PCNA and DDB1/CUL4Acomplexes were found to physically interact with p53 tumor suppressorand its regulator MDM2/HDM2” (Banks, Gavrilova (2006) Cell Cycle.5:1719-1729). Nag et al have also described a physical interactioninvolving p53 and MDM2 as, “Cul4A functions as an E3 ligase andparticipates in the proteolysis of several regulatory proteins throughthe ubiquitin-proteasome pathway. Here, we show that Cul4A associateswith MDM2 and p53” (Nag et al (2004) Cancer Res. 64:8152-8155).

Desired read-out from the bead-based assay for modulators of p53ubiquitination. Where screening compounds results in apositive-screening hit, that is, where there is more AF488 fluorescence,this means that an ACTIVATOR has been discovered. And where screeningcompounds results in a positive-screening hit, where there is aREDUCTION in fluorescence, this means that an INHIBITOR has beendiscovered. A compound that inhibits ubiquitination of p53, suggeststhat the compound can be used for treating cancer. Also a compound thatspecifically inhibits ubiquitination of p53, that is, where the compounddoes not inhibit ubiquitination of other proteins, or where the compoundinhibits ubiquitination of other proteins with inhibition that is lesssevere than for p53, also suggests that the compound can be used fortreating cancer.

Materials. Materials included E3 Ligase kit K-200B from Boston Biochem.Boston Biochem catalog describes this kit as: Mdm2/HDM2 Ubiquitin LigaseKit—p53 Substrate. The following concerns Mdm2, which is part of thiskit. This kit does not include cereblon. Lenalidomide and similarcompounds can bind to either cereblon or to Mdm2, where the end-resultis activation of ubiquitin ligase. Materials also included Diamond WhiteGlass microscope slides, 25 mm×75 mm (Globe Scientific, Paramus, N.J.).Coming Stirrer/Hot Plate (settings from zero to ten) 698 Watts, ModelPC-420. N-hydroxy-succinimide (NHS). Methyltetrazine (mTET).AlexaFluor488 (AF488) (ThermoFisher Scientific). TentaGel beads M NH₂(cat. No. M30102) (Rapp Polymere GmbH). Parafilm (Sigma-Aldrich, St.Louis, Mo.). FIG. 8 shows the structure of Alexa Fluor® 488. Thestructure of Alexa Fluor 488 (AF488) is shown in Product Information forAlexaFluor488-Nanogold-Streptavidin (Nanoprobes, Inc., Yaphank, N.Y.).

(XIII) Cell-Based Assays for Chemical Compounds

Cell-based assays that are conducted in a picowell can use human cells,non-human cells, human cancer cells, non-human cancer cells, bacterialcells, cells of a parasite such as plasmodium cells. Also, cell-basedassays can be conducted with human cells or non-human cells that are“killed but metabolically active,” that is, where their genome has beencross-linked to allow metabolism but to prevent cell division (see, U.S.Pat. Publ. No. 2007/0207170 of Dubensky, which is incorporated herein byreference in its entirety). Moreover, cell-based assays can be conductedon apoptotic cells, necrotic cells, or on dead cells. Cell-based assayswith bacterial cells can be used to screen for antibiotics. Human cellsthat are infected with a virus can be used to screen for anti-viralagents. Combinations of cells are provided for cell-based assays. Forexample, combinations of dendritic cells and T cells are provided toscreen for and identify compounds that stimulate antigen presentationor, alternatively, that impair antigen presentation.

Cell-based assays can be based on a primary culture of cells, forexample, as obtained from a biopsy of normal tissue, a biopsy from asolid tumor, or from a hematological cancer, or from a circulating solidtumor cells. Also, cell-based assays can be based on cells that havebeen passaged one or more times.

Cell-based assays that are conducted in a picowell can use a culturethat contains only one cell, or that contains two cells, three cells,four cells, five cells, or about 2 cells, about 3 cells, about 4 cells,about 5 cells, or a plurality of cells, or less than 3 cells, less than4 cells, less than 5 cells, and so on.

Applicants have conducted working tests based on the followingtechnology. This describes cell-based assays for screening compound forthe exemplary embodiment where lenalidomide (test compound) inhibitsubiquitin-mediated proteolysis of a transcription factor. Thetranscription factors include Ikaros and Aiolos.

The present disclosure provides a cell-based assay that screenscompounds on a bead-bound compounds, and where screening is done with aplate bearing many picowells. The components of the cell-based assayinclude, a picowell for holding a bead-bound chemical library, whereeach bead has attached to it substantially only one, uniform type ofcompound. The compounds are released by way of a cleavable linker.Mammalian cells are cultured in the picowell. The picowell also includesculture medium. The presently disclosed non-limiting example withlenalidomide is a proof-of-principle example that can be used forscreening chemical libraries in order to discover other compounds thatmodulate ubiquitination of a given target protein.

Shorter description of a cell-based assay. Recombinant cells are used asa reagent for detecting and screening for compounds that induceproteolysis of green fluorescent protein (GFP), where the read-out thatidentifies a positively screening compound is the situation wheregreen-colored cells become colorless cells, or cells with reduced greencolor. Regarding the mechanism of this cell-based assay, the mechanismof action of lenalidomide in causing green-colored cells becomecolorless cells, or cells with a reduced green color, is that thelenalidomide binds to a protein called, “cereblon.” In the cell,cereblon is part of a complex of proteins called, “E3 ubiquitin ligase.”Cereblon is the direct target of the anti-cancer drugs, lenalidomide,thalidomide, and pomalidomide. The normal and constitutive activity ofE3 ubiquitin ligase, and its relation to cereblon, has been describedas, “cereblon . . . promotes proteosomal degradation [of targetproteins] by engaging the . . . E3 ubiquitin ligase” (see, Akuffo et al(2018) J. Biol. Chem. 293:6187-6200). In contrast to the normal activityof E3 ubiquitin ligase, when a drug such as lenalidomide, thalidomide,or pomalidomide is added, the result is that the, “lenalidomide,thalidomide, and pomalidomide . . . promote[s] the ubiquitination anddegradation of . . . substrates by an E3 ubiquitin ligase . . . each ofthese drugs induces degradation of transcription factors, IKZF1 andIKZF3” (Kronke et al (2015) Nature. 523:183-188).

Regarding terminology, cereblon has been described as being part of acomplex of proteins that is called, “E3 ligase” and also called, “E3ubiquitin ligase.” Generally, cereblon by itself is not called an “E3ligase. The following excerpts reveal how the word “cereblon” is used.According to Akuffo et al (2018) J. Biol. Chem. 293:6187-6200, “Uponbinding to thalidomide . . . the E3 ligase substrate receptor cereblon .. . promotes proteosomal destruction [of the substrate] by engaging theDDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase.” Consistently, Yang et al(2018) J. Biol. Chem. 293:10141-10157, discloses that, “Cereblon . . .functions as a substrate receptor of the cullin-4 RING E3 ligase tomediate protein [the substrate] ubiquitination.” Zhu et al (2014) Blood.124:536-545, state that, “Thalidomide binds CRBN [cereblon] to alter thefunction of the E3 ubiquitin ligase complex . . . composed of CRBN,DDB1, and CUL4.” Lopez-Girona et al (2012) Leukemia. 26:2326-2335, statethat, “studies identified E3 ligase protein cereblon (CRBN) as a directmolecular target . . . of thalidomide . . . CRBN and . . . DDB1 form afunctional E3 ligase complex with Cul4A and Roc1.”

To view the big picture of the cell-based assay devised and used by theApplicants, the first step is that lenalidomide is added to cells. Thelast step is that IKZF1 and IKZF3 are degraded. Where IKZF1 occurs as afusion protein with GFP, then the last step is that the entire fusionprotein is degraded by the proteasome. Similarly, where IKZF3 occurs asa fusion protein with GFP, then the final step is that this entirefusion protein gets degraded by the proteasome. The result of GFPdegradation is that the cell, which was once green-fluorescing cell, isturned into a non-fluorescing cell.

Longer description of a cell-based assay. This concerns names ofproteins of E3 ubiquitin ligase (a complex of proteins), names ofproteins that bind to this complex, and names of proteins that are thetarget of this complex. For these names, the published literature is notconsistent. Sometimes it refers to the protein by the name of theprotein, and sometimes it refers to the protein using the name of thegene that encodes the protein. For this reason, the following accountuses the protein name together with the gene name, such as “cereblom”(name of protein” and “CRBN” (name of gene). Also, “Ikaros” is the nameof a protein, while the gene's name is IKZF1. Also, “Aiolos” is the nameof a protein, IKZF3 is the name of the gene. “Cullin-ring fingerligase-4” is the name of a protein, and the gene's name is CRL4.“Regulator of cullin-1” is the name of a protein, and the gene's name isROC1. ROC1 is also known as, RBX1 (Jia and Sun (2009) Cell Division.4:16. DOI:10.1186. “Cullin-4A” is the name of a protein and the gene'sname is CUL4A. See, Schafer, Ye, Chopra (2018) Ann. Rheum. Dis.DOI:10.1136, Chen, Peng, Hu (2015) Scientific Reports. 5:10667,Matyskiela et al (2016) Nature. 535:252-257, Akuffo et al (2018) J.Biol. Chem. 293:6187-6200).

E3 ubiquitin ligase catalyzes the transfer of a residue of ubiquitin toa target protein, where the consequence is that the target protein getssent to the proteasome for degradation. The E3 ligase catalyzesattachment of ubiquitin to one or more lysine residues of the targetprotein. Humans express about 617 different E3 ubiquitin ligase enzymes(see, Shearer et al (2015) Molecular Cancer Res. 13:1523-1532). E3ubiquitin ligase is a complex of these proteins: DNA damage bindingprotein-1 (DDB1), Cullin-4 (CUL4A or CUL4B), Regulator of Cullins-1(RoC1), and RING Box-domain protein (RBX1). As stated above, RoC1 is thesame protein as RBX1 (see, Jia and Sun (2009) Cell Division. 4:16.DOI:10.1186). When cereblon (CRBN) joins the E3 ubiquitin ligasecomplex, the resulting larger complex is called: CRL4^(CRBN) (Matyskielaet al (2016) Nature. 535:252-257). The term “CRL4” means, “Cullin-4 RINGLigase” (Gandhi et al (2013) Brit. J. Haematol. 164:233-244, Chamberlainet al (2014) Nature Struct. Mol. Biol. 21:803-809). The abovediscrepancies in nomenclature need to be taken into account when readingthe literature of cereblon.

The following are longer versions of the short excerpts disclosed above.Shown below is yet another form of nomenclature, namely, the term:“CRL4^(CRBN) E3 ubiquitin ligase.” The longer account more fullyintegrates the various names and cellular events. “The relation betweencereblon (CRBN) and E3 ubiquitin ligase complex has been described as,“cereblon (CRBN) promotes proteosomal degradation [of target protein] byengaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase” (Akuffo et al(2018) J. Biol. Chem. 293:6187-6200). Regarding anti-cancer drugs,“lenalidomide, thalidomide, and pomalidomide . . . promote theubiquitination and degradation of . . . substrates by an E3 ubiquitinligase. These compounds bind CRBN, the substrate adaptor for theCRL4^(CRBN)E3 ubiquitin ligase . . . each of these drugs inducesdegradation of . . . transcription factors, IKZF1 and IKZF3” (Kronke etal (2015) Nature. 523:183-188).

This concerns cell-based assays where any given microwell, nanowell, orpicowell contains a bead where bead has covalently linked compounds,where the compound is attached via a cleavable linker, and where thewell contains one or more cultured mammalian cells. Responses tocompounds and to drug candidates of the present disclosure can beassessed by way of one or more biomarkers.

Biomarkers include diagnostic biomarkers, biomarkers that predict if agiven patient will respond (get better) to a given drug, and biomarkersthat predict if a given patient will experience unacceptable toxicity toa given drug (Brody, T. (2016) Clinical Trials: Study Design, Endpointsand Biomarkers, Drug Safety, and FDA and ICH Guidelines, 2^(nd) ed.,Elsevier, San Diego, Calif.). The present disclosure makes use of yetanother kind of biomarker, namely, a biomarker that monitors response ofa patient to a given drug, after drug therapy has been initiated. Togive an example, the following concerns the biomarker “peroxiredoxin6(PRDX6) and lung cancer. According to Hughes et al, “PRDX6 levels incell media from . . . cell lines increased . . . after gefitinibtreatment vs. vehicle . . . PRDX6 accumulation over time correlatedpositively with gefitinib sensitivity. Serum PRDX6 levels . . .increased markedly during the first 24 hours of treatment . . . changesin serum PRDX6 during the course of gefitinib treatment . . . offers . .. advantages over imaging-based strategies for monitoring response toanti-EGFR agents.” Please note comment that the biomarker has advantagesover a more direct measure of efficacy of response, namely, use of“imaging” to detect decrease in tumor size and numbers (Hughes et al(2018) Cancer Biomarkers. 22:333-344). Other biomarkers that monitorresponse to anti-cancer drugs include CA125 for monitoring response toplatin therapy for ovarian cancer, and serum HSPB1 for monitoringresponse to chemotherapy with ovarian cancer (see, Rohr et al (2016)Anticancer Res. 36:1015-1022, Stope et al (2016) Anticancer Res.36:3321-3327).

Cytokine expression. Responses can be assessed by measuring expressedcytokines, such as IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha.These particular cytokines can be simultaneously measured using goldnanostructures bearing antibodies that specifically recognize one ofthese cytokines, where detection involves plasmon resonance (Spackova,Wrobel, Homola (2016) Proceedings of the IEEE. 104:2380-2408, Oh et al(2014) ACS Nano. 8:2667-2676). Cytokines expressed by single cells, suchas a single T cell, can be measured by way of fluorescent antibodies, ina device that includes microwells (Zhu, Stybayeva (2009) Anal. Chem.81:8150-8156). The above methods are useful as reagents and methods forthe present disclosure.

In some embodiments, antibodies to cytokines may be attached to thewalls of the picowells, wherein any cytokines released, ordifferentially released, from cells, as a function of drug exposure canbe captured by the antibodies bound to the walls of the picowells. Thecaptured cytokines may be identified by a second set of labeledantibodies. In some embodiments, antibodies for cytokines may beattached to capping beads. The capping beads may then be embedded in acrosslinking hydrogel sheet that may be peeled off and subjected tofurther analysis, for example, via ELISA, mass spectrometer or otheranalytical techniques.

Apoptosis. Real-time data on apoptosis, and early events in apoptosis ofsingle cells can be measured with Surface-Enhanced Raman Spectroscopy(SERS) and with Localized Surface Plasmon Resonance (LSPR) (see,Stojanovic, Schasfoort (2016) Sensing Bio-Sensing Res. 7:48-54, Loo,Lau, Kong (2017) Micromachines. 8:338. DOI:10.3390). Stajanovic, supra,detects release from cells of cytochrome C, EpCam, and CD49e. Loo et al,supra, measures release from cell of cytochrome C, where detectioninvolves a DNA aptamer (this DNA aptamer works like an antibody). Zhouet al detect early apoptosis in single cells using SERS, where what ismeasured is phosphatidyl serine on the cell membrane (see, Zhou, Wang,Yuan (2016) Analyst. 141:4293-4298). In addition to collecting data onapoptosis, SERS can be used for assessing drug activity by collectingdata on stages of mitosis, release of metabolites, expression of abiomolecule bound to the plasma membrane (see, Cialla-May et al (2017)Chem. Soc. Rev. 46:3945-3961). Plasmon resonance can measure proteindenaturation and DNA fragmentation that occurs in apoptosis (see, Kang,Austin, El-Sayed (2014) ACS Nano. 8:4883-4892). Plasmon resonance (SERS)can distinguish between cancer cells and normal cells, by measuring thepercentage of mitotic proteins in the alpha helix form versus in betasheet form (Panikkanvalappil, Hira, El-Sayed (2014) J. Am. Chem. Soc.136:159-15968). The above methods are suitable as reagents and methodsfor the present disclosure.

Apoptosis can also be measured in cultured cells in a method not usingplasmonic resonance, but that instead uses immunocytochemistry usinganti-cleaved caspase-3 antibody (Shih et al (2017) Mol. Cancer Ther.16:1212-1223).

General information on cell-based assays. Cell-based assays of thepresent disclosure can be used to test responses from human cancercells, cells from a solid tumor, cells from a hematological cancer,human stem cells, human hepatocytes, a pathogenic bacterium, aninfectious bacterium, human cells infected with a bacterium, human cellsinfected with a virus, and so on. The assays can detect morphologicalresponse of the cell, such as migration, as well as genetic responsesand biochemical responses.

Assays of the present disclosure can be designed to detect response ofcells that are situated inside a picowell, or to detect response ofcells that are situated outside a picowell, such as in a nutrient mediumsituated as a layer above the array of picowells. Also, assays of thepresent disclosure can be designed to detect responses of cells, wherecells and beads are situated within a medium, where cells are situatedwithin a medium and beads are above or below the medium, where cells aresituated on top of a medium and where beads are situated above or withinor below the medium.

The present disclosure provides a population of cells to a picowellarray. In embodiments, at least about 5%, at least about 10%, at leastabout 20%, at least about 40%, at least about 60%, at least about 80%,at least about 90%, at least about 95%, or at least about 100%, of thepopulation of cells resides inside the picowells (and not in any regionsituated above the picowells). In embodiments, the proportion of cellsthat resides inside of the wells, with the rest being situated in alayer of nutrient medium residing above the array of wells, can be about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 100%, or in any range defined bytwo of these numbers, such as the range of “about 60% to about 90%.”

Matrix for cells. For assays of biological activity of cells, and wherecells are exposed to compounds released from beads, or where cells areexposed to bead-bound compounds, suitable matrices include those thatinclude one or more of the following: poly-D-lysine (PDL), poly-L-lysine(PLL), poly-L-ornithine (PLO), vitronectin, osteopontin, collagen,peptides that contain RGD sequence, polypeptides that contain RGDsequence, laminin, laminin/fibronectin complex, laminin/entactincomplex, and so on. Suitable matrices also include products availablefrom Corning, Inc., such as, PuraMatrix® Peptide Hydrogel®, Cell-Tak®cell and tissue adhesive, Matrigel®, and so on. See, Corning LifeSciences (2015) Corning Cell Culture Surfaces, Tewksbury, Mass. (20pages), De Castro, Orive, Pedraz (2005) J. Microencapsul. 22:303-315. Inexclusionary embodiments, the present disclosure can exclude anycomposition or method that includes one of the above matrices or one ofthe above polymers.

In embodiments, the present disclosure provides an array, whereindividual picowells contain a bead, one or more cells, and either asolution (without any matrix) or a matrix or a combined solution andmatrix. The matrix can be a hydrogel, polylysine, vitronectin,MatriGel®, and so on.

Activity of bead-bound compounds or of bead-released compounds can beconducted. Assays to assess activity can include, activating orinhibiting an enzyme, activating or inhibiting a cell-signaling cascadeor an individual cell-signaling protein, binding to an antibody (or to acomplementary determining region (CDR) of an antibody, to a variableregion of an antibody), inhibiting the binding of a ligand or substrateto an enzyme (or to an antibody, or to a variable region of anantibody).

For the above assays, the readout can be determined with fluorescenceassays, for example, involving a fluorophore linked to a quencher (F-Q).The linker can be designed to be cleavable by an endoprotease, DNAse,RNAse, or phosopholipase (see, Stefflova, Zheng (2007) FrontiersBioscience. 12:4709-4721). The term “molecular beacon” refers to thistype of F-Q molecule, however, “molecular probe” has also been used torefer to constructs where separation of F and Q is induced byhybridization, as in TaqMan® assays (Tyagi and Kramer (1996) NatureBiotechnol. 14:303-308, Tsourkas, Behlke, Bao (2003) Nucleic Acids Res.15:1319-1330).

Transcriptional profiling in response to drug exposure. The DNA barcodesof this disclosure may be modified to contain response-capture elements,where the response capture elements capture the response of cells toperturbations encoded by the encoding portions of the barcode. In someembodiments, the DNA barcodes may terminate in a poly-T section(multiple repeats of the thymidine nucleotide), where the poly-Tsequence may be used to capture poly-A terminated mRNA moleculesreleased from lysed cells. In some embodiments, the response-capturesequence may be complementary to genes of interest, thereby capturingthe expression profile of desired genes via hybridization to the beadsof this embodiment. In some embodiments, picowells may contain a singlecell picowell whose transcriptional profile is captured on the bead. Insome other embodiments, a plurality of cells may be contained in thepicowell whose transcriptional profile is being captured.

In one exemplary workflow, the following procedure may be followed tocapture transcriptional response of cells to drugs. (a) Picowellsdesigned to capture single cells per well are provided. (b) Acompound-laden, DNA barcoded bead is introduced into the picowells, suchthat one bead is present per picowell. (c) Compounds are released fromthe beads in each picowell by appropriate methods (UV treatment forcompounds attached via UV cleavable linker, diffusion in case of beadssoaked in compounds, acid cleavable, base cleavable, temperaturecleavable etc., as appropriate for the beads of the embodiment). (d) Thepicowells may be isolated from each other via a capping bead thatretains contents within the picowell or by other means such as an airbarrier or an oil barrier on top of the picowells. (e) The cells in thepicowells are allowed to incubate in the presence of the compoundsreleased from the beads for a duration. (f) After a suitable amount oftime, say 1 hr, 2 hrs, 5 hrs, 9 hrs, 12 hrs, 15 hrs, 18 hrs, one day, 3days, one week, two weeks, one months, or another appropriate time basedon the assay, the cells are lysed by a lysing method. The lysing methodsmay involve addition of detergents, repeated cycles of freezing andthawing, heating, addition of membrane disrupting peptides, mechanicalagitation or other suitable means. (g) once lysed, the contents of thecell are exposed to the bead within the picowell, at which time theresponse-capture elements on the beads of the picowell are enabled tocapture the response they are designed for. In some embodiments, theresponse capture are poly-T sequences which capture the complete mRNAprofile of the cell (or cells) within each picowell. In someembodiments, the response-capture elements are designed to capturespecific DNA or RNA sequences from the cell. In some embodiments, thetranscriptional response of the cell may be captured as a function ofdosage (or concentration) of compounds.

(XIV) Perturbation-Response Analysis on Cells

The methods described herein may include a library of perturbations anda library of cells. In some embodiments, the perturbations and the cellsare incubated in a confined environment. During or after the incubation,a barcode identifying the perturbation (“perturbation barcode”) may betransferred to the cells it is incubated with. The method may furtherinclude releasing (i.e., separating or removing) the cells from theperturbation, and subjecting the cells to a second confinement where thecellular content is captured, along with the perturbation barcode. Insome embodiments, the second confinement may contain a cell-specificbarcode, which may be used to subsequently studying the cellular contentand the perturbation barcode, thereby relating the perturbation to thecellular response.

In some embodiments, the methods described herein may include tworeactions, a perturbation reaction that is encoded by a perturbationbarcode and a measurement reaction. In the perturbation reaction, thecells may be subjected to a perturbation. In the measurement reaction, acellular response as a result of the perturbation may be measured. Insome embodiments, the measurement reaction may include a measurementbarcode. In other embodiments, the methods may include carrying over theperturbation barcode to the measurement reaction, and capturing both themeasurement barcode and the perturbation barcode contained in themeasurement reaction, thereby relating the perturbation to the cellularresponse.

Methods described herein may include a perturbation reaction that isencoded by a perturbation barcode. The perturbation reaction includessubjecting the cells to a perturbation and subsequently measuring thecellular response of the cells to the subjected perturbation. Theperturbation barcode may be decoded, either before or after measuringthe cellular response, thus, relating the identity of the perturbationto the measured cellular response.

In some embodiments, the methods include providing a DNA-encoded,bead-bound compound library, wherein the compounds may be released fromthe beads, contacting the DNA-encoded, bead-bound compound library witha library of cells, wherein the contacting may be performed by confininga bead with one or more cells in a first confined volume, releasing thecompound from the bead, and incubating the compound with the cells inthe first confined volume. In some embodiments, either simultaneously orafter the incubation, the DNA barcodes that identify the compounds maybe released from the beads and attached to the cells. The cells with theDNA barcodes attached may then be released from the first confinedvolume and confined again in a second confined volume, wherein thesecond confined volume has reagents to lyse the cells and mechanisms tocapture cellular content and the bead-specific barcode carried by thecells. In some embodiments, the mechanisms used to capture the cellularcontent and the barcodes may involve using a capture barcode, which mayfunction to uniquely identify single cells or a small cluster of cells.In some embodiments, the capture barcode and the bead-specific barcodemay be linked. In some embodiments, all of the confined volumescontaining individually barcoded materials may be merged to create apool of barcoded cellular content and barcodes that are specific to thecells and the bead. The pool of barcoded materials may be analyzed bysequencing to study individual cellular response and link them to theperturbations that caused the cellular response.

In some embodiments, the methods include capturing individual cellswithin droplets. The cells may contain a nucleic acid barcode on theircell membrane to uniquely identify the perturbations experienced by thecells. The cells may be lysed within the droplets and the mRNAs of thecells and the cell-membrane bound nucleic acid barcodes may be capturedon a set of barcoded capture oligonucleotides (“capture barcodes”). Eachdroplet may include a different uniquely barcoded captureoligonucleotides, wherein the barcodes within one droplet have a stretchof substantially identical sequence. The mRNA and cell-membrane boundbarcodes may be copied onto the droplet-barcoded oligonucleotides usinga reverse transcriptase. The nucleic acid materials may be pooledtogether from the droplets by rupturing the droplets. All of the nucleicacid materials may be sequenced to study the transcriptional profile ofthe individual cells and relating it to the perturbations associatedwith the transcriptional profile.

The methods described herein may include subjecting a library of cellsto two barcoded confinements, a perturbation confinement and a lysisconfinement. The cells may be confined individually or as smallclusters. The barcode may include the barcode introduced to the cellswhile perturbing the cells and the barcode introduced while lysing thecells. In some embodiments, the perturbation barcode may be carried bythe cell to the lysis step. In some embodiments, the lysis barcode maybe applied to the cellular content and/or the perturbation barcode,resulting in an establishment of barcoded cellular content that relatesthe cellular content to the perturbation experienced by the cell. Insome embodiments, the perturbation beads may also containresponse-capture probes. In this case, instead of twocompartmentalization steps, a single picowell compartmentalization stepsmay suffice. In such embodiments, the compound barcode may befunctionalized to be capable of capturing cellular responses. In someembodiments, the perturbation barcodes end in a poly(T) segment to whichthe poly(A) tail of the mRNA molecules may hybridize.

In some embodiments, the workflow for single-cell perturbation-responseanalysis is as follows: (1) provide functionalized perturbation beadswherein the perturbation barcodes end in a capture sequence, wherein thecapture sequence may comprise poly(T) nucleotides for the capture ofmRNA, or a set of other appropriate capture probes to capture othercellular responses, (2) capture a library of cells in a picowell array,(3) capture a library of functionalized perturbation into the samepicowells, wherein, in some embodiments, a single cell and a singlefunctionalized bead are captured per well, and in other embodiments, acluster of cells may be captured in a picowell, (4) optionally cover thepicowells with an oil medium to prevent cross contamination of reagentsbetween wells, (5) release the compounds from the perturbation beads andincubate the cells in each well with the compound released from theperturbation beads, (6) lyse the cells within the picowells by flowingin a lysis buffer over the picowells, (7) capture the mRNA or othercellular responses directly on to the tips of the perturbation barcodes,(8) use a polymerase or a reverse transcriptase to copy the cellularresponse onto the perturbation barcode, (9) release the beads from thepicowells by sonication and then cleave the extended perturbationbarcodes from the released beads, or simply cleave the perturbationbarcodes off the beads while the beads are still within the picowells,and (10) subject the cleaved nucleotides (extended perturbationbarcodes) to appropriate library preparation method and sequence thenucleotides so prepared. In some embodiments, the sequenced nucleotidescontain two segments: a perturbation barcode identifying theperturbation/compound that the cell was subjected to, and a responsesegment corresponding to the mRNA expression of the cell that wassubject to the perturbation/compound identified by the perturbationbarcode. This workflow is indicated in FIG. 36 , with optional imagingsteps for QC of the processes. The reverse transcriptase method mayserve to extend the captured RNA onto the bead-attached DNA, therebytransferring cellular content information onto the functionalized beads.The beads can then be pooled, extracted, and analyzed on a sequencer. Insome embodiments, DNA from single cells may also be captured to specificprimers on the functionalized beads. In such embodiments, a polymerasemay replace the reverse transcriptase.

In some embodiments, the perturbation and the cellular-response capturemay happen in two different confinements, as described in FIG. 37 . Theperturbation barcode may be transferred onto the cell surface beforebeing subjected to the cellular-response-capture confinement. Thecellular-response-capture may also involve capturing the perturbationbarcodes carried on the cell-surface, thereby directly relating thecellular response to the perturbation that the cell was exposed to. Insome embodiments, the capture of cellular response can be accomplishedby a Drop-seq method. In some embodiments, the capture of cellularresponse may occur in any commercial single-cell analysis instrumentsuch as a 10× Genomics single-cell instrument, a Raindance single-cellanalysis protocol, a BioRad single-cell isolation instrument, a MissionBio single-cell analysis protocol, a GigaGen instrument and protocol,and/or any other commercially available single-cell analysis instrumentor service.

In some embodiment, cell suspensions may be used as the starting pointfor cells that undergo confinement with perturbation beads. Methods tosuspend cells in aqueous medium or culture cells in suspension arewell-known for a skilled artisan in the field. Methods to suspend cellsand culture cells in suspension are also described in the art. Forexample, in some embodiments, spheroid cell cultures may be used as thestarting point for perturbations, as they capture more cell-cellinteraction signatures than single cells in isolation (see, e.g.,Edmondson et al., Assay Drug Dev Technol. 12:207-218, 2014, Fennema etal., Trends Biotechnol. 31:108-115, 2013, Han et al., Sci Reports5:11891, 2015, Zanoni et al., Sci Reports 6:19103, 2016, the disclosuresof which are all incorporated herein by reference in their entireties).In some embodiments, organoids may be used instead of single cells toundergo high-throughput perturbations (see, e.g., Foley, Nat Methods14:559-562, 2017, Liu et al., Front Pharmacol. 7:334, 2016, Neugebaueret al., BioRxiv April 2017, Skardal et al., Drug Discov Today21:1399-1411, 2016, Boehnke et al., J Biomol Screen 21:931-941, 2016,the disclosures of which are all incorporated herein by reference intheir entireties).

In some embodiments, the cells are obtained from disease-models. Themethods described herein allow massively high-throughput screening ofcompounds across disease-model cells to see if a curative response isobtained by exposure to one or more of the compounds in theperturbation/compound library. In other embodiments, the cells arehealthy cell of various lineage. The methods described herein allowmassively high-throughput mapping of cellular responses to variouscompounds. In some embodiments, the data collected by unbiased screeningof combinatorial compound libraries on cells allows de novo drugprediction based on the known map of drug-cell interactions.

In some embodiments, the confinement used in methods described hereincomprises droplet confinement. In some embodiments, the dropletscomprise aqueous droplets in an oil matrix. In some embodiments, thedroplets are generated in a microfluidic junction comprising mixing ofan aqueous phase and an oil phase. In some embodiments, the microfluidicjunction comprises cells, perturbation beads, and an oil phase. Oneembodiment of microfluidic architectures for creating droplets withcells and beads is illustrated by the “Drop-Seq” method (see, e.g.,Macosko et al., Cell 161:1202-1214, 2015).

In some embodiments, the methods include a hydrogel confinement. In someembodiments, the confinement used in methods described herein compriseshydrogel confinement, wherein cells and beads are embedded in a hydrogelmatrix preventing their free diffusion. In some embodiments, thecolocalization of cells and beads to close proximity to each otheroccurs by chance. In some embodiments, the beads contain bindingmoieties that adhere to the cells, wherein the cell-bead duplex is thenembedded in a hydrogel. In some embodiments, the proximity of a bead toa cell ensures the compounds released from that bead perturb only thatcell without diffusion induced cross reactivity (the cell spacing isfarther than the diffusion radius of the compounds in the hydrogel). Insome embodiments, after the perturbation, the cells are lysed by passinga lysis buffer through the hydrogel, wherein the released cellularcontent are captured on the beads proximal to the cells. Some relevantpublications are: Zhu and Yang, Acc. Chem. Res. 50:22, 2017, Sung andShuler, Lab Chip 9:1385-1394, 2009, Gurski et al., Biomaterials 30:6076,2009, Microfluidic Immunophenotyping Assay Platform for Immunomonitoringof Subpopulations of Immune Cells, pages 1761-1763, 17th InternationalConference on Miniaturized Systems for Chemistry and Life Sciences,MicroTAS, 2013, and US Patent Publication No. US20030175824 A1, thedisclosure of which are all incorporated herein by reference in theirentireties).

In some embodiments, the confinement used in methods described hereincomprises a picowell confinement, wherein individual beads and cells arecaptured within a microfabricated picowell and the assays may beperformed in an array of picowells. A detailed procedure for loadingcells and beads into an array of picowells is described in, e.g., Yuanand Sims, Sci Rep. 6:33883, 2016. In some embodiments, the perturbationbeads also contain response-capture probes, wherein instead of twocompartmentalization steps, a single picowell compartmentalization stepsuffices. In such embodiments, the perturbation barcode mayfunctionalized to be capable of capturing a cellular response. In someembodiments, the perturbation barcodes end in a poly(T) segment to whichthe poly(A) tail of the mRNA molecules may hybridize

In some embodiments, the workflow for single-cell perturbation-responseanalysis is as follows (see, e.g., FIG. 36 ): (1) provide functionalizedperturbation beads wherein the perturbation barcodes end in a capturesequence, wherein the capture sequence may comprise poly(T) nucleotidesfor the capture of mRNA, or a set of other appropriate capture probes tocapture other cellular responses, (2) capture a library of cells in apicowell array, (3) capture a library of functionalized perturbationinto the same picowells, wherein, in some embodiments, a single cell anda single functionalized bead are captured per well, and in otherembodiments, a cluster of cells may be captured in a picowell, (4)optionally cover the picowells with an oil medium to prevent crosscontamination of reagents between wells, (5) release the compounds fromthe perturbation beads and incubate the cells in each well with thecompound released from the perturbation beads, (6) lyse the cells withinthe picowells by flowing in a lysis buffer over the picowells, (7)capture the mRNA or other cellular responses directly on to the tips ofthe perturbation barcodes, (8) use a polymerase or reverse transcriptaseto copy the cellular response onto the perturbation barcode, (9) releasethe beads from the picowells by sonication and then cleave the extendedperturbation barcodes from the released beads, or simply cleave theperturbation barcodes off the beads while the beads are still within thepicowells, and (10) subject the cleaved nucleotides (extendedperturbation barcodes) to appropriate library preparation method andsequence the nucleotides so prepared. In some embodiments, the sequencednucleotides contain two segments: a perturbation barcode identifying theperturbation/compound that the cell was subjected to, and a responsesegment corresponding to the mRNA expression of the cell that wassubject to the perturbation/compound identified by the perturbationbarcode.

In some embodiments, the cellular response is measured optically as amorphological response. In some embodiments, the cellular response ismeasured through labeling of certain cellular features to studydifferences in measured signal after perturbation. In some embodiments,the cellular response is a response engineered into the cells, wherein afavorable stimulation causes the cells to express the engineeredresponse. In some embodiments the engineered response is a reportergene. In some embodiments, the engineered response is the expression ofa fluorescent protein.

In some embodiments, the cellular response comprises the transcriptomeof the cells. In some embodiments, the transcriptional response ismeasured by capturing the mRNA content of the cells and analyzingexpression levels of the mRNA transcripts (see, e.g., Bacher et al.,Genome Biol 17:63, 2016, Svensson et al., Nat Methods 14:381, 2017, Miaoand Zhang, Quantitative Biol 4:243, 2016, the disclosures of which areall incorporated herein by reference in their entireties). In someembodiment, the cellular response comprises the expression and/orpost-translational activation state of proteins or enzymes in the cells.In some embodiments, capturing the cellular response comprises capturingpoly(A) mRNA from the cells using poly(T) oligonucleotides. In someembodiments, the cellular response comprises expression levels ofenhancer RNA in the cells (see, e.g., Rahman et al., Nucleic Acid Res.45:3017, 2017, the disclosure of which is incorporated herein byreference in its entirety). In some embodiments, the cellular responsecomprises the levels of nascent transcripts in the cell. In someembodiments, the nascent transcriptional response is capture by GlobalRun-On Sequencing (GRO-Seq) (see, e.g., Gardini, Meth Mol Biol1468:111-120, 2017 and Danko et al., Nat Methods 12:433, 2015, thedisclosures of which are incorporated herein by reference in theirentireties). In some embodiments, the cellular response comprisesprotein concentrations, wherein the proteins are identified byDNA-tagged antibodies, wherein further the appropriate tag istransferred onto the beads. In some embodiments of the methods describedherein, the cellular response may be captured by imaging, genomicanalysis, or any other tools in molecular biology and sequencing.

EXAMPLES Example 1. First Workflow

The present disclosure provides methods, including that outlined belowas “First Workflow” and as “Second Workflow.”

The First Workflow includes the steps: (1) Generate DELB, (2) Beads intopicowells, (3) Load assay reagents into picowells, (4) Releasebead-bound compounds, (5) Measure assay readout, (6) Rank the assayreadout, and (7) Generate a new set of DELBs.

Generate DELB. First, create the DNA encoded library on beads (DELB).Each bead contains a population of the exact, same compound, thoughslight departures from this may occur where some of the manufacturedcompounds had incomplete couplings or were suffered chemical damage,such as inadvertent oxidation.

Beads into picowells. Then, deposit beads in picowells. In a preferredembodiment, each picowell gets only one bead. Each picowell can have around upper edge, a round lower edge, a solid circular bottom, an opentop, and a wall. The wall's bottom is defined by the round upper edgeand by the round lower edge. In a preferred embodiment, the wall isangled, where the diameter of the round upper edge is greater than thediameter of the round lower edge. In this way, the wall (viewed byitself) resembles a slice of an inverted cone. The picowell array can beprepared, so that there is a redundancy of beads. In other words, thearray can be prepared so that two of the beads, out of the manythousands of beads that are placed into the picowells, contain exactlythe same compound. The redundancy can be, e.g., 2 beads, 3 beads, 4beads, 5 beads, 10 beads, 20 beads, 40 beads, 60 beads, 80 beads, 100beads, and so on, or about 2, about 3, about 4, about 10, about 20,about 40, about 60, about 80, about 100, about 200, about 500, about1,000 beads, and so on, or more than 2, more than 5, more than 10, morethan 20, more than 40, more than 60, more than 80, more than 100, morethan 200, more than 500, more than 1,000 beads, and so on.

Load assay reagents into picowells. Introduce reagents into eachpicowell that can be used to assess biochemical activity of eachbead-bound compound. The biochemical activity can take the form of abinding activity, enzyme inhibition activity, enzyme activationactivity, activity of a living mammalian cell (where the moleculartarget is not known), activity of a living mammalian cell (where themolecular target is known), and so on. The reagent can take the form ofa FRET reagent plus an enzyme. The FRET reagent can be a fluorophorelinked by way of a protease substrate to a quencher. The enzyme can be asubstrate of that protease, which is cleavable by the protease. Thebead-bound compound is being tested for ability to inhibit the protease.

After loading assay materials, each picowell can be capped by a film, ormany or all of the picowells can be capped by one film, or many or allof the picowells can be capped by a film with pimples where each pimplefits into a picowell, or where each picowell is fitted with a poroussphere. In embodiments, about 5% of the volume about 10% of the volume,about 20% of the volume, about 30% of the volume, or about 40% of thevolume of the sphere fits into the picowell (where the remainder isflush with the surface or resides above the surface). In embodiments,about 5%, about 10%, about 20%, about 40%, about 60%, about 80%, about90%, or about 100% of the pimple fits into the picowell.

Release bead-bound compounds. Perform a step that causes release of thebead-bound compound. In embodiments, the step can cause release of about0.1%, about 0.2%, about 0.1%, about 0.2%, about 2%, about 5%, about 10%,about 20%, about 40%, about 60%, about 80%, about 99%, or about 100% ofthe compounds that are attached to a given bead. Release can be effectedby light, by a chemical reagent, by an enzyme, by a shift intemperature, by any combination thereof, and so on.

Release can take the form of: (i) Single release, (ii) Multiple release,(iii) Continual release. Multiple release, for example, can take theform of several emissions of ultraviolet light, where each emission issufficient to cleave about 10% of the bead-bound compound that happensto be attached to the bead at the start of that light emission.Continual release, for example, can take the form of continual emissionof light over the course of one hour, resulting in a steadily increasingconcentrations of free compound. In this situation, the steadilyincreasing concentrations of free compound (cleaved compound) may be forthe purpose of titrating the target of that compound. A titrationexperiment of this kind can be used to assess potency of a givencompound. To provide non-limiting examples, with a single releasemethod, a period of light exposure is followed by a subsequent periodwhere readout is taken, and with a continual release method, lightexposure continues during some, most, or all of the period where readoutis taken.

In exclusionary embodiments, the present disclosure can exclude anymethod, reagent, composition, or system that uses single release, thatuses multiple release, or that uses continual release.

Measure assay readout. Detect the above-disclosed biochemical activity,and the influence of the released compound on that activity. Thisbiochemical activity can take the form of enzymatic activity, activityof a reporter gene, genetic activity (e.g., rate of transcription ortranslation), binding activity (e.g., antigen to antibody), cellularactivity (e.g., change in migration, change in cell-signaling pathway,change in morphology). Activity can be detected by fluorescence,chromogenic activity, luminescence, light microscopy, TaqMan® assays,molecular beacons, mass spectrometry, Raman spectroscopy, LocalizedSurface Plasmon Resonance (LSPR), Surface Plasmon-Coupled Emission(SPCE), Surface-Enhanced Raman Scattering (SERS), and so on. Detectioncan be with methods that are totally remote, such as fluorescencedetection or light microscopy or, alternatively, by methods that involvetaking a sample from the picowell. In one embodiment, a sample thatcontains a mixture of reactants and products can be withdrawn foranalysis by way of a spherical porous sponge that is partially insertedinto one of the picowells.

Rank the assay readout. In this step, assay readouts from a plurality ofdifferent compounds (each type of compound associated with oneparticular bead), are ranked in terms of their ability to activate,inhibit, or in some way to modulate the biochemical activity.

Generate a new set of DELBs. The steps that are described above informthe user of various compounds that exhibit a biochemical activity. Theinformation may take the form of one compound with maximal activity,with the rest having about half maximal activity or less. Alternatively,the information may take the form of several compounds having a similarmaximal activity, with the other compounds having about half maximalactivity or less. A new set of DELBs can be created as follows. One ormore of the highest-ranking compounds (the lead compounds) can be usedas a basis for manufacturing a new set of DELBs, based on one or more ofthe following non-limiting strategies: (i) Replacing an aliphatic chainwith a homolog, such as replacing a propanol side chain with a butanolside chain, (ii) Replacing an aliphatic chain with an isomer, such asreplacing a propanol side chain with an isopropanol side chain, (iii)Replacing a peptide bond with an analog of a peptide bond, such as witha bond that cannot be hydrolyzed by peptidases, (iv) Replacing one typeof charged group with another type of charged group, such as replacing aphosphate group with a phosphonate, sulfate, sulfonate, or carboxylgroup.

Example 2. Second Workflow

The Second Workflow involves picowells that are sealed with caps. Thecaps can take the form of spheres of slightly greater diameter than thediameter of the picowells, where this diameter is measured at the toprim of the picowell (not measured at the bottom of the picowell). Thecap can be made to fit snuggly into the top of the picowell bysubjecting the entire picowell plate to mild-gravity centrifugation. InSecond Workflow, the caps take the form of beads that contain linkers,where each linker is linked to a compound. The linkers are cleavablelinkers, where cleavage released the compounds and allows them todiffuse to the cells. This type of cap is called an “active cap.” TheSecond Workflow includes the steps, (1) Generate DELB, (2) Load assayreagents into picowells, (3) Cap picowells with DELB, (4) Releasebead-bound compounds from the bead that acts as a cap, (5) Measure assayreadout, (6) Determine sequence of the DNA barcode that is on the bead,(7) Rank the assay readout, and (8) Generate a new set of DELBs.

Example 3. Release Control

This concerns controlling and monitoring release of bead-boundcompounds. Applicants devised the following procedure for synthesizingbead-bound release-monitor. See, FIG. 11 and the following text.

FIG. 11 describes steps in the organic synthesis of the above exemplaryembodiment of a bead-bound release-monitor.

Step 1. Provide the Resin

TentaGel® resin (M30102, 10 μm NH2, 0.23 mmol/g, 10 mg, MB160230, 160 μmRAM, 0.46 mmol/g, 2 mg) was weighed into a tube (1.5 mL Eppendorf) andswelled (400 μL, DMA).

Resin was transferred into fritted spin-column (MoBiCol® spin column,Fisher Scientific), solvent removed through filter by vacuum, andpendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400 μL,2×10 min at 40° C.). The MoBiCol spin column has a 10 micrometer largefrit and a luer-lock cap.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

Step 2. Couple Lysine Linker to Resin

A solution was prepared containing L-Fmoc-Lys(Mtt)-OH (21 μmoles, 6.6eq.), DIEA (42 μmoles, 13.3 eq.), COMU (21 μmoles, 6.6 eq.) mixed in DMA(350 μL), incubated (1 min, RT), then added to dry resin inside thefritted spin-column, vortexed, and incubated (15 min, 40° C.) to amidatethe free amine. Resin was filtered by vacuum, and this reaction wasrepeated, once.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

Step 3. Remove the Fmoc Protecting Group

The pendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400μL, 2×10 min at 40° C.).

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

Step 4. Couple the Quencher

A solution was prepared containing QSY7-NHS (4.9 μmoles, 1.55 eq.),Oxyma (9.5 eq, 3.3 eq.), DIC (21 μmoles, 6.6 eq.), TMP (3.5 μmoles, 1.1eq.) mixed in DMA (350 μL), incubated (1 min, RT), then added to dryresin inside the fritted spin-column, vortexed, and incubated (14 hr,40° C.) to amidate the free amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

A solution was prepared containing Acetic Anhydride (80 μmoles, 25.3eq.), TMP (80 μmoles, 25.3 eq.), mixed in DMA (400 μL), mixed then addedto dry resin inside the fritted spin-column, vortexed, and incubated (20min, RT)

Resin was filtered over vacuum, washed (2×DMA, 400 μL, 3×DCM), andincubated in DCM (1 hr, RT), then filtered over vacuum and dried invacuum chamber (30 min, 2.5 PSI)

Step 5. Remove the Mtt Protecting Group

Mtt deprotection cocktail was prepared containing TFA (96 μL), Methanol(16 μL), mixed in DCM (1488 μL) giving 6:1:93% of TFA:Methanol:DCMsolution.

Mtt deprotection cocktail was added to the fully dried resin (400 μL),mixed, eluted by filtration over vacuum, then sequential aliquots of Mttdeprotection cocktail (4×400 μL) were added, mixed, incubated (5 min,RT), and eluted for a combined total incubation time of 20 min at RT.

Resin was filtered over vacuum, and washed (3×DCM, 400 μL, 1×DMA, 400μL, 1×DMA with 2% DIEA, 400 μL, 3×DMA, 400 μL).

Step 6. Couple the Photocleavable Linker to Epsilon-Amino of Lysine

A solution was prepared containing Fmoc-PCL-OH (32 μmoles, 10 eq.),Oxyma (32 μmoles, 10 eq.), DIC (50 μmoles, 15.8 eq.), TMP (32 μmoles, 10eq.) mixed in DMA (400 μL), incubated (1 min, RT), then added to dryresin inside the fritted spin-column, vortexed, and incubated (14 hr,40° C.) to amidate the free F-amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

Step 7. Remove the Fmoc Protecting Group from the Previously CoupledPhotocleavable Linker

The pendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400μL, 2×10 min at 40° C.).

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 1×DMA, 400 μL).

Step 8. Couple the Fluorophore

A solution was prepared containing TAMRA (6 μmoles, 1.9 eq.), TMP (24μmoles, 7.6 eq.), COMU (16 μmoles, 5 eq.), mixed in DMA (400 μL),incubated (1 min, RT), then added to dry resin inside the frittedspin-column, vortexed, and incubated with mixing (2 hr, 40° C., 800 RPM)to amidate the free amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL, 3×DCM, 400μL, 2×DMA, 400 μL, 2×DMSO), then incubated with mixing in DMSO (16 hr,40° C.).

The following provides a broader account of the above-disclosedlaboratory procedures.

Bi-functional linker attached to bead. Bi-functional linker wassynthesized in solution and attached to an amine-functionalized beads.FIG. 11 discloses pathway of organic synthesis, starting with lysine.Lysine-Boc was than connected by TCO linker. The main part of the linkerwas took the form of polyethylene glycol (PEG) with a nitrogen at oneend. Boc was a leaving group in this connecting reaction. The TCA thatwas used was actually a racemate of hydroxy-TCO. The hydroxyl group ofthis TCO derivative was connected to a carbon atom located four carbonatoms away from one side of the double bond (this is the same thing asbeing located three carbon atoms away from the other side of the doublebond). As shown in FIG. 11 , the first product in the multi-stepsynthesis took the form of Boc-lysine-linker-TCO. The hydroxyl groupthat was once part of hydroxy-TCO is still attached to the TCO group,where it is situated in between the aminated-polyethylene glycol groupand the TCO group (FIG. 11 ).

The second set in the synthetic pathway involved treatment with HCl andaddition of a photocleavable linker (PCL). The product of this secondstep was the same as the product of the first step, except with the Bocgroup replaced with the photocleavable linker. The lysine moiety takes acentral position in the product of the second step. Regarding the lysinemoiety, this lysine moiety has a free carboxyl group, and in the thirdstep of the procedure, an aminated bead is connected to this freehydroxyl group, resulting in the synthesis of a bead-bound reagent,where the reagent takes the form of two branches, and where at the endof one branch is a TCO tag, and where at the end of the other branch isan aromatic ring bearing a cleavable bond. To attached a chemicalmonomer to the distal end of the photocleavable linker, first the Fmocgroup is removed, and here the Fmoc group is replaced with a hydrogenatom.

Removing Fmoc. According to Isidro-Llobet et al, “Fmoc . . . is removedby bases mainly secondary amines, because they are better at capturingthe dibenzofulvene generated during the removal” (Isidro-Llobet et al(2009) Chem. Rev. 109:2455-2504). Alternatively, Fmoc can be removed bycatalytic hydrogenolysis with Pd/BaSO₄, or by liquid ammonia andmorpholine or piperidine.

Removal of Fmoc group followed by attaching a chemical monomer.Applicants then condensed a chemical monomer having a carboxylic acidgroup, where the result was generation of an amide bond.

Example 4. Cereblon-Based Assay for Active Compounds

Results from cell-based assays of compounds (cereblon-based assay).Reagents and methods for cell-based assay. Applicants used CCL-2 HeLacells obtained from ATCC (American Type Culture Collection, Manassas,Va.). Cell medium was Gibco DMEM high glucose medium buffered withHEPES. Atmosphere above cell culture was atmospheric air supplementedwith 5% carbon dioxide, with the incubator at 37 degrees C. Cell mediumwas DMEM plus 10% fetal bovine serum, supplemented with GlutaMAX® (GibcoThermofisher), and also supplemented with non-essential amino acids andpenicillin plus streptomycin (Gibco Thermofisher, Waltham, Mass.). HeLacells were transfected with a construct taking the form ofLTR-CTCF-Promoter-IKZF1 (or IKZF3)-mNeon-P2A-mScar-LTR-CTCF. mScarlet isan element used as a positive control. mScarlet encodes red fluorescentprotein called, “mScarlet” (see, Bindels et al (2017) Nature Methods.14:53-56). The promoter is doxycycline inducible promoter, which enablesrapid onset induction and titration of the substrate. P2A is an elementsituated in between two other polypeptides. P2A functions, duringtranslation, to product two separate polypeptides, thus allowing themScar polypeptide to function as a positive control that produces redlight, without being influenced by ubiquitination and degradation of thefusion protein consisting of IKZF1/Green Fluorescent Protein (GFP).mNeonGreen is derived from the lancelet Branchiostoma lanceolatummultimeric yellow fluorescence protein (Allele Biotechnology, San Diego,Calif.). P2A is a region that allows self-cleaving at a point in the P2Aprotein. More accurately, the P2A peptide causes ribosomes to skip thesynthesis of the glycyl-prolyl peptide bond at the C-terminus of a 2Apeptide, leading to the cleavage between a 2A peptide and its immediatedownstream peptide (Kim, Lee, Li, Choi (2011) PLoS ONE. 6:e18556 (8pages).

Demonstration of efficacy of cell-based assay for test compounds. Thefollowing demonstrates use of a cell-based assay for test compoundstaking the form of lenalidomide and analogues of lenalidomide. FIG. 5discloses results from HeLa cells that were transfected with lentiviralvector, where the vector expressed Green Fluorescent Protein (GFP) and ared fluorescent protein (mScarlet). Increasing the concentration ofadded lenalidomide resulted in progressively less green fluorescence,and elimination of green fluorescence at highest concentrations. Butlenalidomide did not substantially decrease red fluorescence. Top:Expression of IKZF1/GFP fusion protein. Bottom: Expression of mScarlettcontrol. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.

FIG. 6 discloses results from HeLa cells that were transfected withlentiviral vector, where the vector expressed Green Fluorescent Protein(GFP) and red fluorescent protein (mScarlet). Increasing concentrationof added lenalidomide resulted in progressively less green fluorescence,and elimination of green fluorescence at highest concentrations. Butlenalidomide did not substantially decrease red fluorescence. Top:Expression of IKZF3/GFP fusion protein. Bottom: Expression of mScarlettcontrol. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.

To summarize the pathway where lenalidomide causes proteolysis of thefusion proteins, first lenalidomide is added to the HeLa cells. Then,the lenalidomide binds to the cereblon that naturally occurs in thesecells. This cereblon occurs in a complex with E3 ubiquitin ligase. E3ubiquitin ligase responds to the lenalidomide by tagging the recombinantIKZF1 fusion protein (or the recombinant IKZF3 fusion protein) withubiquitin. The end-result is that the ubiquitin-tagged fusion protein isdegraded in the cell's proteasome.

Coating the picowell plates. This describes solutions that are appliedto the top surface of a picowell plate, but that do not necessarilyenter and coat inside of picowells. This is also about solutions thatare applied to the top surface of a picowell plate and that enter thepicowells, and that coat the bottom surface of the picowells. Applicantsadded a solution of Pluronic® 127 (Sigma Aldrich, St. Louis, Mo.) to dryplastic. The result is a surface that is hydrophilic, and no longerhydrophobic. Then, the surface was washed with water. Then, phosphatebuffered saline (PBS) was added, where this PBS enters inside thepicowells. Moving air is applied by way of a vacuum, where the result isthat it causes small bubbles in the picowells to expand, and where thebubbles are then replaced with the PBS, and where the end result is thatmuch of the picowell gets filled with PBS. Then, PBS was replaced withvitronectin coating solution (AF-VMB-220) (PeproTech, Rocky Hill, N.J.).Pluronics® 127 is: H(OCH₂CH₂)_(x) (OCH₂CHCH₃)_(y) (OCH₂CH₂)_(z)OH. Afterapplying the vitronectin coating solution, Applicants incubated for 30min at 37 degrees C. to allow the coating solution to get intopicowells. The Pluronic 127 coats the ridges that separate thepicowells, and the vitronectin is at bottom of picowells. HeLa cellsattach to vitronectin and when they attach to the vitronectin, theyadhere to the bottom of the picowell.

HeLa cells were screened for successfully transfected cells by way offlow cytometry. Two criteria were used simultaneously for determiningsuccessful transfection. First, lenalidomide was added to cell media 2days before sorting by flow cytometry. A positive cell was that whichwas red-plus and green-minus, where red-PLUS meant that the cells weretransfected with the gene encoding mScar, and where green-MINUS meantthat the lenalidomide had in fact promoted the ubiquitination anddegradation of the fusion protein, IKZF1/mNeon (or the fusion protein,IKZF3/mNeon). Regarding doxycycline, doxycycline was used at 3micromolar in order to induce expression of the lentiviral vectorconstruct. A concentration/induction curve with doxycycline is shown byGo and Ho (2002) J. Gene Medicine. 4:258-270). After transfection withthe lentivirus vector, the following condition was used to keep IKZF1minimally expressed in growing cells. The condition was to leavedoxycycline out of the medium, and also to use “insulating sequences” inthe construct. The insulating sequences prevent read-through frompromoters outside of the construct. Insulating sequences have beendescribed (see, Anton et al (2005) Cancer Gene Therapy. 12:640-646, Carret al (2017) PLoS ONE. 12:e0176013). Insulating sequences preventpromoters that are outside of the construct from driving expression ofan open reading frame (ORF) that is part of the construct. To put cellsinto picowells, cells can be transferred to the top surface of apicowell plate, at a given ratio of, [number of cells]/[number ofpicowells]. The ratio can be, for example, about 1 cell/40 wells, about1 cell/20 wells, about 1 cell/10 wells, about 2 cells/10 wells, about 4cells/10 wells, about 8 cells/10 wells, about 16 cells/10 wells, about32 cells/10 wells, about 50 cells/10 wells, about 100 cells/10 wells,and so on. The cells can be used for assays in picowells as soon ascells attach to the vitronectin that coats the bottom of the picowell.

Details of lentivirus construct and cell culture. This concernsonstructing reporter cell lines for IKZF1/3, culturing them inpicowells, and assaying them with bulk lenalidomide. The plasmidscarrying reporter construct were assembled from parts using Gibsonassembly (see maps attached). Lentivirus with reporter construct, aswell as UbC driven rtTA-M2.2 were made in LentiX HEK293T cells(Clontech, Palo Alto, Calif.) with 3^(rd) generation packaging system(chimeric CMV promoter and no tat protein). The plasmids weretransfected via calcium precipitation method. Virus supernatant washarvested in the recommended LentiX media plus 1% bovine serum albumin(BSA), and filtered through 0.45 um low protein bind filters(Millipore). The host HeLa cells were obtained from ATCC, cultured instandard conditions. Viral supernatant was applied to sub-confluent HeLaculture, after 24 hours changed to LentiX media with Doxicyclin. Twodays before clone selection, lenalidomide was added to the culture.Clones were selected via fluorescence activated cell sorting (FACS),gated on both AlexaFluor 488 (negative) and Cy3 channels (positive).Clones were grown for 10 days without lenalidomide before assays. Themost stable expression level clones are used for screening.

This describes experiment to seal cells with beads and lyse cellsthrough porous beads. 96 well plate with picowell patterned bottom(MuWells) is treated with Pluronic F127 detergent (Sigma-Aldrich, St.Louis, Mo.) without vacuum applied to passivate upper part of the wells.After 30 min incubation, excess of detergent is washed away withphosphate buffered saline (PBS) or distilled H₂O. Wells are flushed withethanol and dried in the biosafety cabinet with the air flow. Wells arewetted with PBS under strong vacuum to a completion, and PBS is replacedwith Virtonectin coating reagent (Preprotech). The plate is incubatedfor 30 min at 37 C. Vitronectin coating reagent is removed and reportercells are seeded at desirable density. From the moment of cell seeding,media stays in the dish throughout the assay. TentaGel® beads carryingthe photocleavable compound could be seeded before vitronectin coating,or after cell seeding. PEG polymer beads are loaded on top of theculture in the excess over the well number. Spin the plate at 400rcf for1 min. Photo-release the compound off the beads using 365 nm LED lightsource for appropriate amount of time. Incubate in the CO₂ incubatoruntil the imaging (readout of the fluorescent reporters).

Constructs. FIG. 20 and FIG. 21 disclose the relevant constructs. Eachof these figures discloses the sequence that is to be integrated intothe HeLa cell genome, and each of the figures discloses the carriersequence (the sequence belonging to lentivirus). Sequence belonging tolentivirus is from about one o'clock to about nine o'clock, where thissequenced is bracketed by two long terminal repeats (LTRs). Sequencefrom about nine o'clock to about one o'clock gets integrated into HeLacell genome. In detail, first a plasmid is transfected into producercells (HEK93T) (Clontech, Palo Alto, Calif.). The producer cells produceand then release lentivirus. The released lentivirus then infects HeLacells and integrates nucleic acids into the HeLa cell genome.

Optics. For the present cell culture experiments, Applicants used EBQ100Isolated mercury lamp connected to HBO 100 (Carl Zeiss Microscopy, GmbH,Germany), which was connected to an Axiovert 200-M Carl Zeiss microscopewith Ludl Electronic Products stage (Ludl Electronic Products, Ltd.,Hawthorne, N.Y.). Applicants also used filter cubes with mercury lamp,where filter cubes controlled wavelength of excitation and alsocontrolled wavelength of detecting emission. Images were captured withBasler ACA2440-35UM (Basler AG, 22926, Ahrensburg, Germany). Halogenlamp was used, as an alternative to mercury lamp. Microwell plates,picowell plates, and the like, were held in place with a plate holderand an “XY stage” with controller. XY stages and other precisepositioning stages for optics use are available from, Newmark Systems,Inc., Rancho Santa Margarita, Calif., Aerotech, Inc., Pittsburgh, Pa.,Physik Instrumente GmBH, 76228 Karlsruhe, Germany.

Example 5. Mdm2-Based Assay for Active Compounds

Modifying glass to contain an amino group. Silica substrates can bemodified to contain an amino group, by way of one or more of a number of“functional silanes.” These “functional silanes” are3-aminopropyl-triethoxysilane (APTES), 3-aminopropyl-trimethoxysilane(APTMS), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES),N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTMS), andN-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES). Reactions of thesereagents with glass can be conducted in a vapor phase or in a solutionphase (see, Zhu, Lerum, Chen (2012) Langmuir. 28:416-423).

Results from biochemical assays of compounds (MDM2-based assay).Laboratory methods. The following reagent was applied to a glass slide.The glass slide was modified to have amino groups. The reagent wasNHS-PEG-mTET. NHS is N-hydroxy-succinimide. NHS is a type of activatedester. NHS is useful in bioconjugation reactions, such as surfaceactivation of microbeads or of microarray slides (Klykov and Weller(2015) Analytical Methods. 7:6443-6448).

PEG is polyethylene glycol. mTET is methyltetrazine. This reagent wasmixed with DMSO, and then a volume of 2 microliters was applied to theglass slide. The mixture was made by mixing 10 microliters of 50 mMNHS-PEG-mTET with 30 microliters DMSO. The NHS group reacts with theamino groups of the glass side, where the result is that the mTET groupis affixed to the glass slide. The goal of the mTET was to create acovalent link between the slide and the bead.

TCO and tetrazine can mediate “click chemistry” reactions. Examples ofthese click chemistry reactions, is using antibodies that arefunctionalized with tetrazine to couple with DNA that is functionalizedby TCO. Or using antibodies modified with TCO to couple withtetrazine-modified beads (see, van Buggenum et al (2016) ScientificReports. 6:22675 (DOI:10.1038), Rahim et al (2015) Bioconjug. Chem.18:352-360, Haun et al (2010) Nature Nanotechnol. 5:660-665).

In detail, the glass slide was prepared by applying a sheet of parafilmto the top of the slide, where the parafilm had an aperture cut out ofthe middle, where the drop of the above mixture was applied in theaperture directly to the glass slide. Before applying the mixture, theglass slide with the parafilm on top was heated at full heat for 90seconds, in order to create a tight seal between the parafilm and theslide, in order to prevent seepage of liquids after applying the mixtureto the open area (the aperture) in the parafilm. The glass slide, withthe 2 microliter droplet sitting in the aperture cut into the Parafilm,was incubated overnight at room temperature. During the incubation, theglass slide was inside a petri dish, where the dish was covered with aglass cover that covered the top and sides of the petri dish. Before theovernight incubation, a square of Parafilm was placed over the drop andover the surrounding Parafilm, in order to prevent water fromevaporating from the drop.

Inventive method to make complex of slide/bead/antibody. Applicants'method used beads that were functionalized by TCO. The TCO groups of thebead mediated covalent attachment of the methyltetrazine-functionalizedslide to the bead. Also, the TCO groups of the bead mediated covalentattachment of the methyltetrazine-functionalized anti-p53 antibody tothe bead.

Applicants surprisingly found that, if the first step is to contactslide and bead, then subsequent addition of antibody will NOT result incovalent attachment of the antibody to the bead. Also, Applicantssurprisingly found that, if the first step is to contact bead withantibody, then subsequent transfer of this mixture to the slide will NOTresult in covalent attachment of the bead to the slide. In a preferredmethod, all of these three reagents—the slide, the bead, and theantibody—are simultaneously brought into contact with each other. Inanother preferred embodiment, the bead and antibody are first mixedtogether to initiate covalent linking of the bead to the antibody, andthen immediately or within a few minutes, this mixture is applied to theslide, where the result is covalent linking of the bead to the slide.

Nature of the enzyme-based screening assay. The assay takes the form ofa glass slide with an attached bead. The bead contains attachedantibodies that are specific for binding to the transcription factor,p53. This antibody can bind to human p53 and also to ubiquitinated humanp53. So far, it can be seen that the assay method involves a sandwichbetween the following reagents:

Slide/Covalently bound bead/Bead-bound anti-p53 Ab/Ubiquitinated p53

The readout from this assay is ubiquitinated-p53, where theubiquitinated-p53 is detected by a fluorescent antibody that is specificfor ubiquitin. In detail, the antibody is a polyclonal antibody made inthe goat, where the antibody is tagged with a fluorophore (AF488). FIG.8 discloses the structure of AF488. This fluorescent antibody binds toubiquitin. Thus, when ubiquitinated-p53 is detected, what exists is thefollowing sandwich:

Slide/Covalently bound bead/Bead-bound bound anti-p53 Ab/Ubiquitinatedp53/Fluorescent Ab

Example 6. Sequencing DNA in Picowells

Sequencing of bead-bound DNA barcodes was performed, where beads weresituated in a picowell, one bead per picowell. The assay method involvedinterrogating each position on the bead-bound DNA barcode, one at atime, by way of transient binding of fluorescent nucleotides. Each beadcontained about one hundred attomoles of coupled DNA barcode, wherecoupling was by click-chemistry. This number is equivalent to aboutsixty million oligonucleotides, coupled per bead. For each base on theDNA barcode, the assay involves adding all four fluorescent dNTPs at thesame time. Without implying any limitation, the four fluorescent dNTPswere AF488-dGTP, CY3-dATP, TexasRed-dUTP, and CY5-dCTP. Fluorescentsignals were captured, and then processed by ImageJ software (NationalInstitutes of Health, NIH), to provide a corresponding numerical value.The data are from sequencing five consecutive nucleotides (all in a row)that was part of the bead-bound DNA barcode. The bead-bound DNA barcodeincluded a DNA hairpin region. The bases in the DNA hairpin regionannealed to itself, resulting in the formation of the hairpin, and wherethe 3′-terminal nucleotides in this DNA hairpin served as a sequencingprimer. Sequencing by transient binding was initiated at this3′-terminus. The sequencing assay was performed in triplicate, that is,using three different beads, where one DNA barcode sequence was used foreach of the three beads. In other words, each of the three beads wasexpected to provide a sequencing read-out identical to that provided bythe other two beads.

FIG. 28 discloses sequencing results, where sequencing was conducted onbead-bound DNA barcode. What is shown are results from interrogating thefirst base, the second base, the third base, the fourth base, and thefifth base. For each of these bases, what is separately shown, by way ofseparate histogram bars, is the fluorescent emission produced withinterrogation with AF488-dGTP, CY3-dATP, TexasRed-dUTP, and CY5-dCTP,respectively. Each of the four histogram bars has different graphics:AF488-dGTP (black outline, gray interior), CY3-dATP (black outline,white interior), TexasRed-dUTP (solid black histogram bar), and CY5-dCTP(solid gray histogram bar). The bead diameter was 10-14 micrometers,after swelling in aqueous solution. The volume of the picowell was 12picoliters.

The template sequence that was interrogated was:5′-CTCACATCCCATTTTCGCTTTAGT-3′. For this particular sequencing assay,five consecutive bases were interrogated, where the fluorescent dNTPsthat gave the biggest fluorescent signal were fluorescent dGTP, dATP,dGTP, dUTP, and dGTP, which corresponds to a sequence on the templatethat is dC, dT, dC, dA, and dC. Thus, the sequencing results were 100%accurate. The results demonstrate that the bead-bound DNA barcodes canbe sequenced, that is, when the DNA barcode is still bound to the bead.In other words, the bead-bound DNA barcodes are sequenceable.

Example 7. Cell Barcoding

Introduction to the concept of barcoding. This introduces the concept ofbarcoding. A common barcoding technique is barcoding the transcriptomeof a given single cell. FIG. 36 and FIG. 37 illustrate steps forprocedures where the transcriptome is captured and amplified, inpreparation for future sequencing. FIG. 36 shows lysis of cells torelease mRNAs, followed by reverse transcription. FIG. 37 shows captureof mRNAs by way of immobilized poly(dT), followed by reversetranscription, and finally sequencing. Sequencing can be with NextGeneration Sequencing (NGS).

Some or most of the messenger RNA (mRNA) molecules from a given cell canbe tagged with a common barcode, where this tagging allows theresearchers to determine, for any given mRNA sequence, the origin ofthat coding sequence in terms of a given cell. For example, wherenucleic acids representing each of the separate transcriptomes from onehundred different single cells are mixed together, and where the nucleicacids from each of the 100 different single cell has its own barcode,then the following advantage will result. The advantage is that nucleicacids from all of the transcriptomes can be mixed together in one testtube, and then subjected to Next Generation Sequencing, where thebarcode enables the user to identify which information is from the samecell.

The above advantage is described in a different way, as follows. Inusing mRNA barcoding, a given single cell is processed so thatinformation from some or most of the mRNA molecules from that cell areconverted to corresponding molecules of cDNA, where each of these cDNAmolecules possesses exactly the same DNA barcode. This barcodingprocedure can be repeated with ten, twenty, 100, several hundred, orover 1,000 different cells, where the cDNA molecules from each of thesecells is distinguished by having a unique, cell-specific barcode. Thismethod enables the researcher to conduct DNA sequencing, all in onesequencing run, from a pool of all of the barcoded cDNA molecules fromall of the cells (all barcoded cDNA molecules mixed together, prior tosequencing) (see, Avital, Hashimshony, Yanai (2014) Genome Biology.15:110).

Barcodes that tag nucleic acids compared with barcodes that tag theplasma membrane. Guidance is available for preparing libraries ofchemicals, where each chemical, or where all members of each class ofchemicals, is associated with a unique DNA barcode (see, Brenner andLerner (1992) Proc. Nat'l. Acad. Sci. 89:5381-5383, Bose, Wan, Carr(2015) Genome Biology. 16:120. DOI 10.1186). With the above barcodingexample in mind, the following provides another type of barcoding whichcan also be applied to a particular, single cell. The present disclosureprovides cell-associated barcoding that takes the form of a tag that isstably attached to the cell's plasma membrane.

Option of at least two kinds of barcodes that get attached to the plasmamembrane-bound. A barcode used for tagging the plasma membrane of givencell can include a first barcode that identifies the type of cell, and asecond barcode that identifies a perturbant that was exposed to thecell. For example, the first barcode can identify the cell asoriginating from a healthy human subject, Human Subject No. 38 fromClinical Study No. 7, a human primary colorectal cancer cell line, afive-times passaged human primary colorectal cancer cell line, amultiple myeloma human subject with multiple myeloma, a treatment-naiveHuman Subject No. 23 with multiple myeloma, or from atreatment-experienced Human Subject No. 32 with multiple myeloma.

Also, the barcode can identify a “perturbant” that was given to thatparticular single cell (given either before or after barcoding). The“perturbant” can be an anti-cancer drug, a combination of anti-cancerdrugs, a combinatorially generated compound, or a combination of anantibody drug and a small molecule drug. The barcoding can be used tokeep track of a given single cell, and can be used to correlate thatcell with subsequent behaviors such as activation or inhibition with oneor more cell-signaling pathways, increased or decreased migration,apoptosis, necrosis, change in expression of one or more CD proteins(CD, cluster of differentiation), change in expression of one or moreoncogenes, change in expression of one or more microRNAs (miRNAs).Expression can be in terms of, transcription rate, level of a givenpolypeptide in the cell, change in location of a given protein fromcytosolic to membrane-bound, and so on.

Tagging cell-surface oligosaccharides of membrane-bound glycoproteins.Methods and reagents are available for connecting tags, such as DNAbarcodes, to the plasma membrane of a living cell. Tagging can beaccomplished with a reagent consisting of a covalent complex of a DNAbarcode with a reactive moiety that attacks and covalently binds tooligosaccharide chains of membrane-bound glycoproteins. The literatureestablishes that hydrazide biocytin can be used to connect biotin tocarbohydrates on membrane-bound glycoproteins. The present disclosureuses this reagent, except with the biotin replaced with a DNA barcode.The carbohydrate needs to be oxidized to form aldehydes. The hydrazidereacts with the aldehyde to form a hydrazine link. The sialic acidcomponent on the oligosaccharides is easily oxidized with 1 mM Nameta-periodate (NaIO₄). In conducting the oxidation step, andhydrazide-linking step, buffers with a primary amine group should beavoided. See, for example, “Instructions. EZ-LinkHydrazide Biocytin.Number 28020. ThermoScientific (2016) (4 pages), Bayer (1988) Analyt.Biochem. 170:271-281, Reisfeld (1987) Biochem. Biophys. Res. Commun.142:519-526, Wollscheid, Bibel, Watts (2009) Nature Biotechnol.27:378-386.

Another method for tagging the oligosaccharide moiety of glycoproteinson living cells, is to use periodate oxidation and aniline-catalyzedoxime ligation. This method uses mild periodate oxidation of sialicacids and then ligation with an aminoxy tag in the presence of aniline.In a variation of this method, galactose oxidase can be used tointroduce aldehydes into terminal galactose residues and terminalN-acetylgalactosamine (GalNAc) residues of oligosaccharides. Galactoseoxidase catalyzes the oxidation, at carbon-6, to generate an aldehyde.Following aldehyde generation, one can couple with aminoxybiotin usinganiline-catalyzed ligation (see, Ramya, Cravatt, Paulson (2013)Glycobiology. 23:211-221). The present disclosure replaces the biotinwith a DNA barcode and provides aniline-catalyzed ligation of anaminoxy-DNA barcode.

Tagging mediated by an antibody bound to the cell surface. The presentdisclosure provides methods and reagents for attaching barcodes to theplasma membrane of a cell, where attachment is mediated by an antibodythat specifically binds to a membrane-bound protein. The antibody can becovalently modified with trans-cyclooctene (TCO) where this modificationcan be conducted with an overnight incubation at 4 degrees C. (see,Supporting Information (5 pages) for Devaraj, Haun, Weissleder (2009)Angew. Chem. Intl. 48:7013-7016). This covalent modification of antibodycan be carried out with the reagent, trans-cyclooctene succinimidylcarbonate (Devaraj, Haun, Weissleder (2009) Angew. Chem. Intl.48:7013-7016). The antibody-tetrazine complex can then be contacted witha cell, resulting in membrane-bound antibodies. The membrane-boundantibodies each bear a tetrazine moiety, which enables tagging of theantibody via click chemistry, such as, by exposing the antibodies to aDNA barcode-tetrazine complex.

Tetrazine can be introduced at free amino groups of the antibody, usingthe reagent, N-hydroxysuccinimide ester (NHS) (see, van Buggenum,Gerlach, Mulder (2016) Scientific Reports. 6:22675). Once the antibodycontains one or more tetrazine groups, the antibody can be furthermodified by attaching a DNA barcode, by way of a reagent that is TCO-DNAbarcode. With this modified antibody in hand, the antibody can then beused for a tagging living cell, where the antibody binds to amembrane-bound protein of the cell.

A complex of tetrazine-DNA barcode can be prepared. This complex canthen be introduced into a cell medium, where the medium includes cells,and where the cells bear the attached antibody-TCO complex. Where thetetrazine-DNA barcode contacts the membrane-bound antibody-TCO complex,the result is a click chemistry reaction where the cells become taggedwith the DNA barcode. This click chemistry reaction can be carried outfor 30 minutes at 37 degrees C.

Preferred antibodies for use in the above procedure are those that bindtightly and specifically to membrane-bound proteins of the plasmamembrane, where the membrane-bound protein occurs in high abundance, forexample, at over 50,000 copies per cell membrane, and where themembrane-bound protein is stable on the cell surface and does not muchrecycle into the cell's interior, and where the membrane-bound membranedoes not much shed into the culture medium.

Tagging membrane-bound proteins with azide followed by click chemistrywith an octyne conjugate. Azide can be introduced on membrane-boundproteins of a living cell by way of the enzyme, lipoic acid ligase,followed by attachment of a fluorinated octyne compound that isconjugated to a DNA barcode. The conjugation of a fluorinated octynecompound to a fluorophore is described (see, Jewett and Bertozzi (2010)Chem. Soc. Rev. 39:1272-1279, Fernandez-Suarez, Bertozzi, Ting (2007)Nature Biotechnol. 25:1483-1487). To reiterate, “Ting and co-workersintroduced azides into mammalian cell-surface proteins using . . .lipoic acid ligase . . . [t]he protein could then be labeled with afluorinated cyclooctyne-conjugated fluorescent dye-conjugatedfluorescent dye” (Jewett et al, supra).

Example 7. Caps Over Picowells

Capping picowells. Each picowell was capped with a sphere, one sphere toeach picowell, where the sphere fits into the aperture (top opening) ofthe picowell. To apply the spheres to the picowell plate, the spheresare put into growth media and suspended, then applied to the top surfaceof the picowell plate, and the sphere allowed to settle. Then, theentire plate is placed in a centrifuge and spun at a low-gravity, inorder to get a firm sitting of the spheres in the aperture of eachpicowell.

Active caps and passive caps. FIG. 18A shows an active cap inserted intothe top of a picowell, and FIG. 18B shows a passive cap inserted intothe top of a picowell. Preferably, the caps are made of material that issofter than the material used to make the picowell plate, where theresult is slight deformation of the cap when it is pressed into theaperture of the picowell, and where the result is a snug fit thatprevents leakage. In embodiments, the present disclosure provides one ormore of active caps, passive caps, or both active caps and passive caps.Each cap may be free-standing and not connected to any other cap. In analternative embodiment, to more caps may be connected together, forexample, by way of a sheet of polymer that is capable of being laid uponthe top surface of the plate, and where a plurality of caps protrudefrom the bottom of the sheet of polymer, and where the protruding capsare predeterminedly spaced in order to fit into each picowell. An activecap may be used instead of a bead that is capable of sitting on thefloor of a picowell. The active cap contains many attached copies ofsubstantially identical compounds, where each compound is attached tothe active cap (shown here in the sample of a spherical bead), and wherecleavage results in release of the compounds into the solution thatresides in the picowell (FIG. 18A).

Regarding the passive cap, the passive cap is porous and it acts like asponge. It absorbs products from biochemical reactions, and thusfacilitates collection of products where the goal of the user is todetermine the influence of a given compound on living, biological cellsthat are cultures in the picowell. In other words, the compoundstimulates the cells to respond, where the response takes the form ofincreased (or decreased) expression of one or more metabolites, andwhere some of the metabolites diffuse towards the passive cap and areabsorbed by the passive cap. The user can then collect the passive capsand analyze the metabolites that had absorbed to the passive cap (FIG.18B).

Polymer mat that adheres to an array of caps. FIG. 19 illustrates apolymer mat that is capable of adhering to each cap in an array ofporous caps. Once adhered, the polymer mat can be peeled away andremoved, bringing with it each porous cap in the array. As a result, thepolymer mat with the porous caps can be used for assays that measuremetabolites or other chemicals that are associated with the porous cap.

To provide a step-wise example, each well in an array of many thousandsof picowells can contain one bead, where each bead contains one type ofcompound, where the compound is attached via a cleavable linker. Thepicowell also contains a solution as well as cultured cells. Thepicowell is sealed with a porous cap, and where the porous cap contactsthe solution and is able to capture (sample, absorb, absorb) metabolitesthat are released from the cultured cells. The metabolites can bemetabolites of the compound, or the metabolites can take the form ofcytokines, interleukins, products of intermediary metabolism, microRNAmolecules, exosomes, and so on. Finally, a solution of polyacrylamide ispoured over the picowell plate, and the polyacrylamide allowed to soakinto the thousands of porous caps, and then solidify in the form of amat that is firmly adhered to each and every one of the caps. Thesolified mat is then removed, where each cap is separately analyzed forabsorbed metabolites.

In preferred embodiments, a polyacrylamide gel is used to crosslink thecapping beads into the enmeshing layer or the mat. The protocol tocreate an 20% solution of polyacrylamide solution that can be pouredover the picowell array to cure and enmesh the capping bead is asfollows. Add 4 ml of a 40% bis-acrylamide solution and 2 ml of 1.5 MTris pH 8.8 to 1.8 ml distilled deionized water. Just before pouringthis mixture over the capped picowell array, 80 microliters of the freeradical initializer ammonium persulfate (APS, 10% stock solution), and 8microliter of the free radical stabilizerN,N,N′,N′-tetramethylethylene-diamine (TEMED) are added to begincrosslinking of the gel. The gel layer is poured before completecrosslinking and allowed to fully crosslink over the capped picowellarray. One fully crosslinked (stiff enough to be handled, or roughly 60minutes of setting), the polyacrylamide layer may be peeled off usingtweezers. It is found that the capping beads are lifted off the tops ofthe picowells and get attached to the polyacrylamide layer. Thisbehavior can be observed for multiple bead types includingpolyacrylamide beads, Tentagel beads, polystyrene beads and silicabeads.

Measuring efficacy of cap in preventing leaks. In embodiments, theefficacy of a cap can be determined by using the bead with thephotocleavable linker. Images of a picowell, or of several picowells inone particular picowell array can be captured just before exposingpicowells to UV light, and in the time frame after exposing picowells toUV light. For example, images can be captured at t=minus ten seconds andat t=10 seconds, 20 sec, 40 sec, 60 sec, 2 minutes, 4 min, 8 min, 15min, 60 min, 90 min, 2 hours, 3 hours, and 4 hours. Excellent efficacycan be shown where the fluorescence of a given well at 2 hours is equalto at least 90%, at least 95%, at least 98%, or about 100% thefluorescence found at t=10 seconds, with subtraction of the backgroundimage taken at t=minus ten seconds. Images can also be taken of a regionof the picowell plate outside of the picowell, for example, in theimmediate vicinity of the cap. Excellent efficacy can be shown where thefluorescence of an area on the surface of the plate (outside of thepicowell) and in the immediate vicinity of the cap is less than 1%, lessthan 0.5%, less than 0.1%, less than 0.05%, less than 0.01%, less than0.005%, or less than 0.001%. This comparison may be made without regardto the volume of the fluid in the well, and without regard to the volumeof any fluid situated on top of the plate and outside of the cap, andhere, the comparison may simply take into account the entire visualfield that is captured by the light detector. Alternatively, thecomparison may be made with correction of the depth of the fluid (depthof picowell, depth of fluid on top of the picowell plate). Alsoalternatively, the comparison may take into account diffusion of anyleaking fluorophore over the entire surface of the picowell plate.

How barcoding fits into the reagents and methods of the presentdisclosure. The following provides further embodiments of the reagentsand methods of the present disclosure.

Reagents and capabilities. A microscopic bead is provided. Themicroscopic bead can be covalently modified by a plurality of firstlinkers, each capable of coupling by way of solid-phase synthesis withmonomers, where completion of the solid-phase synthesis creates a memberof a chemical library. This member of the chemical library isbead-bound. The same microscopic bead can be covalently modified by aplurality of second linkers, each capable of being coupled with aplurality of DNA barcodes. This member of the DNA barcode is bead-bound.

Example 9. DNA Barcode of the Present Disclosure

This concerns a set of information that can be printed on paper, orstored in computer language, that provides a “DNA barcode” thatcorrelates a DNA sequence with a chemical library member. This DNAbarcode may be called a “legend” or a “key.” The DNA barcode alsoprovides nucleic acids that can identify a specific class of chemicalcompounds, such as analogs of a specific FDA-approved anti-cancer drugs,or that can identify the user's name, or that can identify a specificdisease that is to be tested with the bead-bound chemical library.

Example 10. Lenalidomide Analogs

FIGS. 13, 14, and 15 disclose the conversion of lenalidomide to threedifferent derivatives, each derivative bearing a carboxylic acid group.Each of these carboxylic acid groups can subsequently be used tocondensed with the bead-linker complex. In this situation, where thecarboxylic acid group is condensed to the bead-linker complex, it isattached at the position that was previously occupied by Fmoc.

Starting with a primary amine and converting it to a carboxylic acid(FIG. 13 ). Applicants take the approach of generating a library ofcompounds by converting a compound with a primary amine to a compoundwith a carboxyl group. FIG. 13 discloses starting with lenalidomide.Lenalidomide has a primary amine. To this is added, succinic anhydridein 4-dimethylaminopyridine (DMA) and acetonitrile (ACN). The succinicanhydride condenses with the primary amino group, resulting inlenalidomide bearing a carboxylic acid group. The term “cat.” in thefigure means, catalytic.

Subsequently, this carboxylic acid group can be linked to a bead. Thus,the resulting complex is: BEAD-succinic acid moiety-lenalidomide.

FIG. 14 discloses starting with linalidomide and addingt-butyl-bromoacetate, to give an intermediate. The intermediate is thentreated with FmocOSu (o-succinimide), to produce a final product that isa carboxylic acid derivative of lenalidomide. The carboxylic acid moietycan then be condensed with a free amino group, for example, with thefree amino group that once had an attached Fmoc group. Alternatively,the carboxylic acid can be condensed with the free amino group of achemical monomer residing on the bead, where the result of thecondensation is two chemical monomers attached to each other.

FIG. 15 discloses lenalidomide as the starting material. Thelenalidomide is reacted with 3-carboxybenzaldehyde, where the aldehydegroup condenses with the amino group, resulting in yet another type ofcarboxylic acid derivative of lenalidoimide.

FIG. 16A, FIG. 16B, and FIG. 16C discloses yet another approach ofApplicants for generating a library of novel and unique bead-boundcompounds, where compounds can be released from the bead, and thentested for activity in cell-based assays or in cell-free assays. Each ofthe three compounds is a lenalidomide analogue, where the primary amineis in a unique position of the benzene ring.

Example 11. Picowells Containing Cells Together with Beads that have aCoupled Response-Capture Element

The present disclosure provides reagents, systems, and methods forassessing response of a cell to a compound, and where response that ismeasured takes the form of changes in the transcriptome. “Changes in thetranscriptome” can refer, without implying any limitation, to change inamount each and every type of unique mRNA in the cell, and well as tochange in amount of a pre-determined set of mRNA molecules in the cell.“Changes in transcriptome” includes change from below the lower limit ofdetection to becoming detectable, as well as change from beingdetectable to dropping below the lower limit of detection, where thesechanges are associated with release of the bead-bound compound.

Cells can be lysed by adding detergent or surfactant to the picowellarray. For example, a volume of buffer containing detergent can bepipetted into a microwell that contains, within it, many thousands ofpicowells. The detergent can be allowed to diffuse into all of thepicowells, causing lysis of the cells within, release of mRNA, andfinally binding by the bead-bound “capture response element.”

Cell lysis. Cells can be lysed by one or more cycles of freezing andthawing (Bose, Wan, Carr (2015) Genome Biology. 16:120. DOI 10.1186).Cells can also be lysed with perfluoro-1-octanol with shaking (Macosko,Basu, Satija (2015) Cell. 161:1202-1214, Ziegenhain (2017) MolecularCell. 65:631-643, Eastburn, Sciambi, Abate (2014) Nucleic Acids Res.42:e128). Also, cells can be lysed by a combination of a surfactant(Tween-20®) and a protease (Eastburn, Sciambi, Abate (2013) Anal. Chem.85:8016-8021). Lysis of cells results in release of mRNA. The mRNA iscaptured by the bead that resides in the same picowell as the lysed cell(or cells). The bead contains a huge number of bead-boundpolynucleotides, where each polynucleotide contains two nucleic acid,where the first nucleic acid contains a common DNA barcode and thesecond nucleic acid contains a “response capture element.” Where thegoal is indiscriminate capture of all mRNAs in the cell, the “responsecapture element” can take the form of poly(dT). This poly(dT) binds tothe poly(A) tail of the mRNA molecules.

More cell lysis conditions. Cell lysis can be effected by exposure todetergent with a sodium salt, for example, 0.05% Triton X-100 with 15 mMNaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, 0.1% Triton X-100with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, 0.2%Triton X-100 with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mMNaCl, or 0.5% Triton X-100 with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75mM NaCl, 100 mM NaCl, or with detergent with a potassium salt, such as,0.05% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl, 100mM KCl, 0.1% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mMKCl, 100 mM KCl, 0.2% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl,75 mM KCl, 100 mM KCl, or 0.5% Triton X-100 with 15 mM KCl, 25 mM KCl,50 mM KCl, 75 mM KCl, 100 mM KCl. Exposure can be for 10 min, 20 min, 40min, or 60 min at about 4 degrees C., or at room temperature (23 degreesC.), and so on.

Present disclosure can assess influence of a compound on an expressionprofiles. A bead-bound capture element can take the form of one or moredeoxyribonucleotides that can specifically hybridize to one or more mRNAmolecules of interest, where the one or more mRNA molecules areassociated with a specific disease. Expression profiles for variousdiseases are available, for example, for colon cancer (Llarena (2009) J.Clin. Oncol. 25:155 (e22182), ovarian cancer (Spentzos (2005) J. Clin.Oncol. 23:7911-7918), and lung adenocarcinoma (Takeuchi (2006) J. Clin.Oncol. 11:1679-1688). To give a similar example, what can also becharacterized is the influence of a released compound on mRNAsassociated with non-hepatic tumor cells that have metastasized to theliver (see, Barshack, Rosenwald, Bronfeld (2008) J. Clin. Oncol. 26:15Suppl. 11026, Barshack (2010) Int. J. Biochem. Cell Biol.42:1355-1362.).

Capturing the transcriptome. Methods are available for capturing mRNA byhybridizing their polyA group to immobilized poly(dT) (see, Dubiley(1997) Nucleic Acids Res. 25:2259-2265, Hamaguchi, Aso, Shimada (1998)Clinical Chem. 44:2256-2263, D. S. Hage (2005) Handbook of AffinityChromatography, 2^(nd) ed, CRC Press, page 549).

After capture of mRNA molecules released from the lysed cell (or cells),the bead-bound polynucleotide serves as a primer that supports reversetranscription from the mRNA, resulting in a bead-bound complementary DNA(cDNA), and where this bead-bound cDNA can be sequenced. Alternatively,the bead-bound cDNA can be released from the bead, where the bead-bound“response capture element” is coupled to the bead with a cleavablelinker, such as with a photocleavable linker. If a photocleavable linkeris used, cleaving conditions for releasing bead-bound compounds(compounds made from a chemical monomer library) but not also cleave thebead-bound “response capture element.”

Where cells are exposed to a bead-bound compound or to a compoundreleased from a bead, cells can be screened for a genetic response, forexample, by characterizing any changes in the transcriptome with orwithout exposure to the compound. Also, cells can be screened for aphenotypic response, for example, apoptosis, change in activity of oneor more cell-signaling proteins, or change in cell-surface expression ofone or more CD proteins. CD is Cluster of Differentiation (See, Lal(2009) Mol. Cell Proteomics. 8:799-804, Belov (2001) Cancer Res.61:4483-4489, IUIS/WHO Subcommittee on CD Nomenclature (1994) Bull.World Health Org. 72:807-808, IUIS-WHO Nobenclature Subcommittee (1984)Bull. World Health Org. 62:809-811). For some phenotypic responseassays, the cells must not be lysed.

The present disclosure addresses the unmet need to partition differentdrugs to different cells, for example, by exposing a single cell to onetype of drug where exposure occurs in a picowell.

The present disclosure also eliminates the need to prepare barcodedmRNA, where mRNA is released from a cell followed by preparing cDNA (inthis type of barcode, all mRNA from a given cell receives the samebarcode, when the transcriptome is converted to corresponding library ofcDNA).

Parameters during cell incubation with the perturbant. For any givencompound or some other type of perturbant, parameters that can be variedor controlled light, temperature, pH of cell medium, sound,concentration and exposure time to a reagent (reagent can be thecompound released from the bead, an enzyme substrate, a cytokine, acompound that is already an established drug, a salt), mechanicagitation, an antibody against a cell-surface protein, and so on.

Barcoding the cell. Cells can be incubated with a bead-bound compound orwith the compound following cleavage from a bead-bound cleavable linker.During or after incubation, cells can be barcoded with a membrane-boundbarcode that identifies the perturbant. This membrane-bound barcode canbe coupled to oligosaccharides of the cell membrane, polypeptides of thecell membrane, or phospholipids of the cell membrane.

Response capture elements other than poly(dT). Messenger RNA can becaptured by way of the 5-prime 7-methylguanosine cap. This method isespecially useful where there polyA tail is short (see, Blower,Jambhekar (2013) PLOS One. 8:e77700). Also, mRNA can be captured usingimmobilized DNA that is specific for a coding region of the mRNA. Thismethod is called, “RNA exome capture,” and variations of this name.According to Cieslik et al, “Unique to capture transcriptomics is anovernight capture reaction (RNA-DNA hybridization) using exon-targetingRNA probes” (Cieslik (2015) Genome Res. 25:1372-1381).

MicroRNA (miRNA). The present disclosure can assess the influence of areleased bead-bound compound on expression profile of miRNAs in a givencell or, alternatively, on expression profiles of the population ofmRNAs that are specifically bound by a given species of miRNA (Jain,Ghosh, Barh (2015) Scientific Reports. 5:12832). For example, thepresent disclosure provides a bead that contains: (1) Bead-boundcompound, (2) Bead-bound DNA barcode, and (3) Bead-bound responsecapture element, where the response capture element either capturesmiRNA or where the response capture element includes a species of miRNA(as part of the response capture element). Expression profiles formicroRNA have been found for various types of cancer, for example,breast cancer breast cancer (Tanja (2009) J. Clin. Oncol. 27:15 Suppl.538).

Methods are available for capturing selected populations of mRNA fromthe entire transcriptome. Selectivity can be conferred by using one typeof microRNA, such as miR-34a, as a bridging compound in a “pull-down”assay. In brief, “The transcripts pulled down with miR-34a were . . .enriched for their roles in growth factor signaling and cell cycleprogression” (Lal, Thomas, Lieberman (2011) PLOS Genetics. 7:e1002363).The mRNA molecules that are captured are those that bind to the miR-34A.

Further methods for capturing mRNA and analyzing expression level isavailable (Bacher (2016) Genome Biology. 17:63, Svensson (2017) NatureMethods. 14:381, Miao and Zhang (2016) Quantitative Biol. 4:243, Gardini(2017) Nature Methods. 12:443). Cellular response taking the form ofchanges in enhancer RNA can be measured (see, Rahman (2017) Nucleic AcidRes. 45:3017).

Transfer Devices

In order to ensure that a single bead is deposited into a single wellwhen the assay conditions require the same, a transfer device isemployed to achieve a single bead per single well. The transfer devicecan be included in a robotic process, a manual process or a combinationof robotic and manual processes. The transfer device can be based onmagnetic or non-magnetic beads. Magnetic beads preferably employ amagnetic transfer device with reversible magnetism. Non-magnetic beadsemploy electrostatic attraction or engineering principles based on sizeand gravity.

Non-magnetic beads used herein can be electrostatically charge due tothe components bound thereto such as DNA which is negatively charged atpH 7. A positively charged resin will interact with the negativelycharged beads and having a binding affinity that can participate in beadpickup. Optionally, a vacuum source can be used in combination with theelectrostatically charged beads and oppositely charged resin. Such acombination can be used robotically to capture a single bead and thenrelease it into a single well. In such an embodiment, a capturingelement such a pipette can be sized with a diameter that is sufficientlywide to hold a single bead. The pipette is fitted with an electrostaticporous resin oppositely charged to the charged beads. A vacuum source isused to assist in retrieval of the beads from a bead source. Multiplebeads may be extracted from the source and into a single pipette.However, only a single bead is contacted with the resin. Afterretrieval, the vacuum is slowly released until a partial vacuum pressureand the electrostatic interaction between the single bead and the resinis sufficient to hold that bead whereas the remaining beads in thepipette fall back into the bead source leaving a single bead in a singlepipette. The robotics of this device can include multiple sets ofpipettes in a single array that can then be positioned over the wells ofthe device. The remaining partial vacuum is removed and an individualbead falls into an individual well.

Alternatively, a dispenser having a plurality of cavities sized to holda single bead is designed so that each cavity aligns with a single wellon an assay device and allows the assay device to reside either over orunder the dispenser. Cavities on the dispenser are filled with beads andthe assay device is fitted over the dispenser and is aligned so thateach cavity corresponds to a single well. The walls of the dispenser andthe assay device form a closed chamber between each cavity and each wellsuch that the bead cannot relocate to another part of either thedispenser or the assay device. Reversing the position of the assaydevice such that it is placed under the dispenser results in transfer ofa single bead into a single well.

Magnetic dispensers can be used with magnetic beads. In one embodiment,the magnetic dispenser can locate a weak magnetic force or a reversiblemagnetic force. In the case of a weak magnetic force, the magnet isfitted into a pipette. A vacuum source is combined with the magnet toretrieve beads into the pipette. The pipette is likely to hold multiplebeads but only one of which contacts the magnet. As the vacuum pressureis reduced, those beads held only by the vacuum are released back intothe bead source leaving only a single bead in a single pipette. Whendispensing the bead into a well of an assay device, the vacuum isremoved and the remaining bead falls into that well.

The following examples provide exemplary embodiments for a device orsystem for providing a single bead to each well of an assay devicehaving a multiplicity of wells. The assay device having a single beadper well can be used in any of the assays described herein.

Example 12: Method of Dispensing a Single Bead into a Single Cavity (ofa Dispenser) and/or a Single Well (of an Assay Device)

A method for perturbing a cell and capturing a response of the cell tothe perturbation according to an exemplary embodiment is provided. Themethod may start with providing a transfer device with at least onecavity configured to be aligned with at least one well of an assaydevice. The assay device and the transfer dispenser may be moved to fitonto or mate with each other. The assay device and the transferdispenser may be fitted onto or mated with each other with or without agap being present between said assay device and said transfer device.The method may include aligning cavities of the transfer dispenser withwells of the assay device. The method may include fitting onto or matingthe assay device and the transfer dispenser with each other to form acontainment space. The method may include releasing a single bead fromthe cavity through said containment space. The method may includedepositing the single bead into said well. Any step of the method mayrepeat. The method may start or end at any point within theabove-referenced sequence.

Example 13: Computer-Implemented Control System

FIG. 38 is a schematic diagram of a computer device or system forperturbing a cell and capturing a response of the cell to theperturbation including at least one processor and a memory storing atleast one program for execution by the at least one processor accordingto an exemplary embodiment. Specifically, FIG. 38 depicts a computerdevice or system 3800 comprising at least one processor 3830 and amemory 3840 storing at least one program 3850 for execution by the atleast one processor 3830. In some embodiments, the device or computersystem 3800 can further comprise a non-transitory computer-readablestorage medium 3860 storing the at least one program 3850 for executionby the at least one processor 3830 of the device or computer system3800. In some embodiments, the device or computer system 3800 canfurther comprise at least one input device 3810, which can be configuredto send or receive information to or from any one of: an external device(not shown), the at least one processor 3830, the memory 3840, thenon-transitory computer-readable storage medium 3860, and at least oneoutput device 3870. The at least one input device 3810 can be configuredto wirelessly send or receive information to or from the external devicevia a means for wireless communication, such as an antenna 3820, atransceiver (not shown) or the like. In some embodiments, the device orcomputer system 3800 can further comprise at least one output device3870, which can be configured to send or receive information to or fromany one from the group consisting of: an external device (not shown),the at least one input device 3810, the at least one processor 3830, thememory 3840, and the non-transitory computer-readable storage medium3860. The at least one output device 3870 can be configured towirelessly send or receive information to or from the external devicevia a means for wireless communication, such as an antenna 3880, atransceiver (not shown) or the like.

In some exemplary embodiments, the computer device or system 3800 isconfigured to send instructions to one or more robotic components formoving one or more of the elements described herein. The presentinvention may be practiced completely automatically with the assistanceor robotic arms or hands, or may be practiced with a combination ofmanual and robotic approaches.

Each of the above identified modules or programs corresponds to a set ofinstructions for performing a function described above. These modulesand programs (i.e., sets of instructions) need not be implemented asseparate software programs, procedures or modules, and thus varioussubsets of these modules may be combined or otherwise re-arranged invarious embodiments. In some embodiments, memory may store a subset ofthe modules and data structures identified above. Furthermore, memorymay store additional modules and data structures not described above.

The illustrated aspects of the disclosure may also be practiced indistributed computing environments where certain tasks are performed byremote processing devices that are linked through a communicationsnetwork. In a distributed computing environment, program modules can belocated in both local and remote memory storage devices.

Moreover, it is to be appreciated that various components describedherein can include electrical circuit(s) that can include components andcircuitry elements of suitable value in order to implement theembodiments of the subject innovation(s). Furthermore, it can beappreciated that many of the various components can be implemented on atleast one integrated circuit (IC) chip. For example, in one embodiment,a set of components can be implemented in a single IC chip. In otherembodiments, at least one of respective components are fabricated orimplemented on separate IC chips.

Example 14: Cavity with Sub-Cavity

FIG. 39 is a side cross-sectional view of a dispenser 3901 with at leastone cavity, and a sub-structure within the cavity according to anexemplary embodiment. The dispenser 3901 is configured to facilitatesize exclusion at a larger end of the spectrum of bead sizes. Thedispenser 3901 is configured to exclude relatively larger beads.Specifically, the dispenser 3901 may include a main cavity and asub-cavity. The main cavity may be recessed into a top surface 3905 ofthe dispenser 3901. The main cavity may have a side wall 3910. The maincavity may have a bottom floor 3915. The bottom floor 3915 may have asub-cavity recessed therein. The sub-cavity may be defined by a sidewall 3920 and may have a sub-cavity floor 3925. The sub-cavity may beconfigured to receive a single bead 10. Although the dispenser 3901 isshown with rectilinear edges, the edges may include any suitablestructure including curved or chamfered edges, and the like.

The cavity or the sub-cavity of the dispenser 3901 may be provided inany suitable size including an opening having a diameter on the order ofpicometers. The dispenser 3901 may be configured so that only onecorrectly-sized bead 10 may be loaded (trapped) into a respectivesub-cavity. Un-trapped beads in the picowells may be washed away byturbulence. The dispenser 3901 could be used as shown; or the dispenser3901 may be flipped over to transfer beads to an assay device.

Example 15: Tapered Cavity

FIG. 40 is a side cross-sectional view of a dispenser 4001 with at leastone cavity, a tapered side wall, an open top end, and an open bottom endaccording to an exemplary embodiment. The dispenser 4001 may includeconical shape cavities. The conical shaped cavities may be configured toreject relatively larger beads by turbulence with a fluid primarilyincident on a top surface of the dispenser 4001. The dispenser 4001 mayinclude a tapered cavity. The tapered cavity may be recessed into a topsurface 4005 of the dispenser 4001. The tapered cavity may have a sidewall 4010. The tapered cavity may have an opening in the bottom thereof.The opening may be recessed into a bottom surface 4025 of the dispenser4001. The tapered cavity may be configured to receive a single bead 10by virtue of a difference in a shape of an upper portion of the taperedcavity relative to a smaller shape of a lower portion of the taperedcavity. Although the dispenser 4001 is shown with rectilinear edges orrelatively sharp corners, the edges may include any suitable structureincluding curved or chamfered edges, and the like.

The tapered cavity of the dispenser 4001 may have a conical shape. Thetapered cavity may be provided in any suitable size including an openinghaving a diameter on the order of picometers.

The dispenser 4001 may be configured so that only one correctly-sizedbead may be loaded (held) in the tapered cavity. The dispenser 4001 maybe configured to permit relatively smaller beads to pass through theopening in the bottom surface 4025 of the dispenser 4001. Relativelylarger beads may be washed away. The dispenser 4001 could be used asshown; or the dispenser 4001 may be flipped over to transfer beads to anassay device.

In one exemplary embodiment, the opening in the bottom surface 4025 mayinstead be closed off to include a floor provided that only one beadfits into one corresponding cavity.

As noted in related U.S. patent application Ser. No. 16/774,871, anoptional gap may be removed according to an exemplary embodiment.Specifically, a dispenser may be integrated or fitted with an assaydevice to deliver beads from cavities to wells of an assay device.Alignment of a dispenser with the assay device can be facilitated withan optional locking mechanism. When locked in place, the dispenser andthe assay device need not be flush against each other. The optional gapcan be present, so long as the gap is smaller than the bead or otherassay components. The fitted dispenser and assay device may be invertedfor delivery of the beads or other assay components from the dispenserinto the wells of the assay device.

The position of the transfer dispenser and the assay device relative toeach other allows dispensing of beads such that a single assay componentis deposited into a single well. Specifically, in one embodiment, thereis provided a dispenser comprising a multiplicity of cavities whereineach cavity is configured to reversibly hold/capture only a single assaycomponent, such as a bead, and further wherein the dispenser isconfigured to fit or mate with an assay device comprising a multiplicityof wells such that, when fitted, each cavity in said dispenser isaligned with a single well in said assay device. Upon release, assaycomponents move from dispenser into assay device such that a singleassay component is deposited into a single well.

Also, the alignment of cavities and wells ensures that a single bead isdeposited into a single well.

Example 16: Cavity with Magnet

FIG. 41 is a side cross-sectional view of a dispenser 4101 with at leastone cavity, and a magnet disposed at the bottom of each cavity accordingto an exemplary embodiment. The dispenser 4101 may be configured suchthat a magnetic force on a bead directly incident on the magnet issufficient to hold the bead and resist turbulent flow of a fluidprimarily incident on a top surface of the dispenser 4101. Also, thedispenser 4101 may be configured such that a magnetic force on a secondbead on top of or adjacent to the bead directly incident on the magnetis insufficient to hold the second bead thus promoting removal of thesecond bead with turbulent flow of the fluid primarily incident on thetop surface of the dispenser 4101.

The dispenser 4101 may include a main cavity. The main cavity may berecessed into a top surface 4105 of the dispenser 4101. The main cavitymay have a side wall 4110. The main cavity may have a bottom floor 4115.The bottom floor 4115 may include a magnet or magnetic surface 4120. Themagnet or magnetic surface 4120 may be configured to form a magneticbond with a single bead 10. Although the dispenser 4101 is shown withrectilinear edges, the edges may include any suitable structureincluding curved or chamfered edges, and the like.

In some exemplary embodiments, the magnet or magnetic surface 4120 maybe flat. Whereas, in other exemplary embodiments, the magnet or magneticsurface 4120 may be concave (as shown in FIG. 41 ), which advantageouslyincreases a surface area for contact between the surface 4120 and thesingle bead 10.

The dispenser 4101 may include a cavity with a relatively small andrelatively weak magnet at the bottom of each cavity. The cavity may beprovided in any suitable size including an opening having a diameter onthe order of picometers. Each of the beads 10 may be magnetic beads. Thedispenser 4101 may be configured so that only one bead may be loaded(held) by the magnet 4120 in the cavity. Beads not held by the magnet4120, or subject to relatively small magnetic forces, could be washedaway. The dispenser 4101 could be used as shown; or the dispenser 4101may be flipped over to transfer beads to an assay device. In aflipped-over state, if gravity alone is not sufficient to dislodge thebead 10, relatively gentle vibration or other gentle mechanical forcemay be imparted to the dispenser 4101 to dislodge the bead 10 from thedispenser 4101.

Example 17: Pipette with Mesh Insert

FIG. 42 is a side cross-sectional view of system 4200 including adispenser 4280, and a pipette 4220 with an optional mesh insert 4255according to an exemplary embodiment. The pipette 4220 may include aconduit, which may couple a valve or pumping mechanism to an end 4250 ofthe pipette 4220. The conduit may have two openings at the opposingends. The shape at the ends may be any suitable shape includingsubstantially circular. The conduit may include a series of tubes andconnectors to provide a sealed pathway of a pressure-reducing pump withthe end 4250. The diameter of conduit may be wider than a diameter of alargest bead 4290 under test. The conduit and/or the end 4250 may bemade of polyvinyl chloride (PVC), clear plastic, Tygon, plexiglass,and/or the like. The conduit and/or the end 4250 may be rigid,semi-rigid, or flexible.

A conduit may be configured to relocate a single bead. The conduit canbe used in any type of device. In various exemplary embodiments, theconduit may be configured: to place a bead in a cavity of a dispenser;and/or to place a bead in a well of an assay device; and/or to remove abead from a cavity of a dispenser; and/or to remove a bead from a wellof an assay device.

In some exemplary embodiments, the conduit and/or the end 4250 may beformed from a polymer. The polymer may be a polyimide, which issufficiently rigid to resist bending or buckling, but flexible enough toflex upon contact with a substrate and avoid breaking the substrate,including a microwell plate. When the conduit and/or the end 4250 isformed of polyimide and with a relatively thin wall thickness, i.e., onthe order of about 8 microns thick, the material may be relativelyfragile. As such, the polyimide conduit and/or end 4250 may be boundwith a protective material, such as polytetrafluoroethylene (PTFE). Forexample, when the polyimide conduit and/or end 4250 has a length of morethan about 45.0 cm, the polyimide conduit and/or end 4250 may beprotected with the protective material such as PTFE.

In an exemplary embodiment, the provision of the conduit and/or the end4250 with a flexible tip permits an operator to pick up beads that are,for example, stuck to walls of a well. The flexible tip permits theoperator to turn the conduit and/or the end 4250 through a radius of thewell. The flexible tip may be very forgiving in that the flexible tipallows the operator to pick up beads without a need to use an enzymesuch as protease. The flexible tip may be configured to stably interfacewith a surface of the well. In some uses, the operator may push theflexible tip against the wall to extract a bead that is stuck to a wallof the well. The relatively rigid and fragile glass pipettes of thedeveloped art do not have such functionality.

The polyimide may be provided in any suitable configuration including asa single layer, multiple layers of similar materials or multiple layersof dissimilar materials.

The flexible tip may be capable of depositing a bead held within theflexible tip. The flexible tip may be capable of withdrawing orextracting a bead held by another device.

In some exemplary embodiments, a thickness of a wall of the conduitand/or the end 4250 may be on the order of about 8 to 450 microns. Insome exemplary embodiments, the conduit and/or the end 4250 may includereinforcement. In some exemplary embodiments, the conduit and/or the end4250 may include a coating such as polytetrafluoroethylene (PTFE), e.g.,TEFLON; however, such coating is not required and may be omitted tomaintain a desirable translucent property, which permits visualizationof a lumen of the conduit and/or the end 4250 and facilitatesverification.

The dispenser 4280 may include a plurality of cavities 4285 a, 4285 b,4285 c . . . 4285 n.

An interior space within the pipette 4220 may include a mesh layer 4255.The mesh layer 4255 may be a mesh filter. In another example, the layer4255 may merely be a bar from a first interior wall of end 4250 to asecond interior wall of end 4250.

The mesh layer 4255 may be a mesh screen stretched the interior space ofthe pipette 4220. The mesh screen may be made out of small fibers,plastic rods, metal wires, and/or the like. The layer 4255 may be aplate extending across the interior space of the pipette 4220. The platemay contain at least one aperture, which may permit excess liquid topass therethrough.

A bead may be picked with the pipette 4220 by vacuum connected to thepipette 4250. The mesh insert 4255 may ensure the bead is held inside atip of the pipette 4220. That is, with the vacuum pulling a bead up, themesh insert 4255 may function as a stop within an opening within thepipette 4220. To transfer the bead, a tip of the pipette 4220 may beplaced on top of the assay device. To release the bead, one may releasethe vacuum force or apply positive pressure to move the bead into theassay device.

In use, the pipette 4220 may encounter more than one bead. Theconfiguration of the pipette 4220 is such a single bead 10 is held bythe mesh insert 4255. Electrostatic forces incident on the single bead10 may permit the pipette 4220 to hold the single bead 10 with orwithout vacuum. A combination of magnetic, vacuum or gravity forces maybe used to control the bead 10 within the pipette 4220.

In some embodiments, the mesh insert 4255 may be comprise a reversiblemagnetic material that allows air to flow through the material.

Example 18: Virtual Wells

In lieu of a physical well or cavity, a virtual well may be provided.Specifically, for example, starting with a planar surface, droplets ofwater and/or test solution may be added onto the planar surface. Wateror test solution containing one or more beads will be relatively heavierthan water or test solution not containing a bead. Turbulence may beutilized to remove relatively light (non-bead-containing) water orsolution from the planar surface thus retaining relatively heavy(bead-containing) water or solution on the planar surface. As the weightof a single bead of an appropriate size and mass disposed in the wateror test solution per unit volume is known, the weight may be monitored.The process may be performed, verified and repeated, as desired, untilthe planar surface has a single bead situated over each virtual well.

The present invention is not to be limited by compositions, reagents,methods, systems, diagnostics, laboratory data, and the like, of thepresent disclosure. Also, the present invention is to not be limited byany preferred embodiments that are disclosed herein.

The subject matter described herein may be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The embodiments set forth in the foregoing description donot represent all embodiments consistent with the subject matterdescribed herein. Instead, they are merely some examples consistent withaspects related to the described subject matter. Although a fewvariations have been described in detail above, other modifications oradditions are possible. In particular, further features and/orvariations may be provided in addition to those set forth herein. Forexample, the embodiments described above may be directed to variouscombinations and subcombinations of the disclosed features and/orcombinations and subcombinations of several further features disclosedabove. In addition, the logic flows depicted in the accompanying figuresand/or described herein do not necessarily require the particular ordershown, or sequential order, to achieve desirable results. Otherembodiments may be within the scope of the following claims.

In particular and in regard to the various functions performed by theabove described components, devices, circuits, systems and the like, theterms used to describe such components are intended to correspond,unless otherwise indicated, to any component which performs thespecified function of the described component (e.g., a functionalequivalent), even though not structurally equivalent to the disclosedstructure, which performs the function in the herein illustratedexemplary aspects of the claimed subject matter. In this regard, it willalso be recognized that the innovation includes a system as well as acomputer-readable storage medium having computer-executable instructionsfor performing the acts and/or events of the various methods of theclaimed subject matter.

The aforementioned systems/circuits/modules have been described withrespect to interaction between several components/blocks. It can beappreciated that such systems/circuits and components/blocks can includethose components or specified sub-components, some of the specifiedcomponents or sub-components, and/or additional components, andaccording to various permutations and combinations of the foregoing.Sub-components can also be implemented as components communicativelycoupled to other components rather than included within parentcomponents (hierarchical). Additionally, it should be noted that atleast one component may be combined into a single component providingaggregate functionality or divided into several separate sub-components,and any at least one middle layer, such as a management layer, may beprovided to communicatively couple to such sub-components in order toprovide integrated functionality. Any components described herein mayalso interact with at least one other component not specificallydescribed herein but known by those of skill in the art.

In addition, while a particular feature of the subject innovation mayhave been disclosed with respect to only one of several implementations,such feature may be combined with at least one other feature of theother implementations as may be desired and advantageous for any givenor particular application. Furthermore, to the extent that the terms“includes,” “including,” “has,” “contains,” variants thereof, and othersimilar words are used in either the detailed description or the claims,these terms are intended to be inclusive in a manner similar to the term“comprising” as an open transition word without precluding anyadditional or other elements.

As used in this application, the terms “component,” “module,” “system,”or the like are generally intended to refer to a computer-relatedentity, either hardware (e.g., a circuit), a combination of hardware andsoftware, software, or an entity related to an operational machine withat least one specific functionality. For example, a component may be,but is not limited to being, a process running on a processor (e.g.,digital signal processor), a processor, an object, an executable, athread of execution, a program, and/or a computer. By way ofillustration, both an application running on a controller and thecontroller can be a component. At least one component may reside withina process and/or thread of execution and a component may be localized onone computer and/or distributed between two or more computers. Further,a “device” can come in the form of specially designed hardware;generalized hardware made specialized by the execution of softwarethereon that enables the hardware to perform specific function; softwarestored on a computer-readable medium; or a combination thereof.

Moreover, the words “example” or “exemplary” are used herein to meanserving as an example, instance, or illustration. Any aspect or designdescribed herein as “exemplary” is not necessarily to be construed aspreferred or advantageous over other aspects or designs. Rather, use ofthe words “example” or “exemplary” is intended to present concepts in aconcrete fashion. As used in this application, the term “or” is intendedto mean an inclusive “or” rather than an exclusive “or”. That is, unlessspecified otherwise, or clear from context, “X employs A or B” isintended to mean any of the natural inclusive permutations. That is, ifX employs A; X employs B; or X employs both A and B, then “X employs Aor B” is satisfied under any of the foregoing instances. In addition,the articles “a” and “an” as used in this application and the appendedclaims should generally be construed to mean “one or more” unlessspecified otherwise or clear from context to be directed to a singularform.

Computing devices typically include a variety of media, which caninclude computer-readable storage media and/or communications media, inwhich these two terms are used herein differently from one another asfollows. Computer-readable storage media can be any available storagemedia that can be accessed by the computer, is typically of anon-transitory nature, and can include both volatile and nonvolatilemedia, removable and non-removable media. By way of example, and notlimitation, computer-readable storage media can be implemented inconnection with any method or technology for storage of information suchas computer-readable instructions, program modules, structured data, orunstructured data. Computer-readable storage media can include, but arenot limited to, RAM, ROM, EEPROM, flash memory or other memorytechnology, CD-ROM, digital versatile disk (DVD) or other optical diskstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or other tangible and/or non-transitorymedia which can be used to store desired information. Computer-readablestorage media can be accessed by at least one local or remote computingdevice, e.g., via access requests, queries or other data retrievalprotocols, for a variety of operations with respect to the informationstored by the medium.

On the other hand, communications media typically embodycomputer-readable instructions, data structures, program modules orother structured or unstructured data in a data signal that can betransitory such as a modulated data signal, e.g., a carrier wave orother transport mechanism, and includes any information delivery ortransport media. The term “modulated data signal” or signals refers to asignal that has at least one of its characteristics set or changed insuch a manner as to encode information in at least one signal. By way ofexample, and not limitation, communication media include wired media,such as a wired network or direct-wired connection, and wireless mediasuch as acoustic, RF, infrared and other wireless media.

In view of the exemplary systems described above, methodologies that maybe implemented in accordance with the described subject matter will bebetter appreciated with reference to the flowcharts of the variousfigures. For simplicity of explanation, the methodologies are depictedand described as a series of acts. However, acts in accordance with thisdisclosure can occur in various orders and/or concurrently, and withother acts not presented and described herein. Furthermore, not allillustrated acts may be required to implement the methodologies inaccordance with the disclosed subject matter. In addition, those skilledin the art will understand and appreciate that the methodologies couldalternatively be represented as a series of interrelated states via astate diagram or events. Additionally, it should be appreciated that themethodologies disclosed in this specification are capable of beingstored on an article of manufacture to facilitate transporting andtransferring such methodologies to computing devices. The term articleof manufacture, as used herein, is intended to encompass a computerprogram accessible from any computer-readable device or storage media.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Although at least one exemplary embodiment is described as using aplurality of units to perform the exemplary process, it is understoodthat the exemplary processes may also be performed by one or pluralityof modules.

The use of the terms “first”, “second”, “third” and so on, herein, areprovided to identify various structures, dimensions or operations,without describing any order, and the structures, dimensions oroperations may be executed in a different order from the stated orderunless a specific order is definitely specified in the context.

Approximating language, as used herein throughout the specification andclaims, may be applied to modify any quantitative representation thatcould permissibly vary without resulting in a change in the basicfunction to which it is related. Accordingly, a value modified by a termor terms, such as “about” and “substantially,” are not to be limited tothe precise value specified. In at least some instances, theapproximating language may correspond to the precision of an instrumentfor measuring the value. Here and throughout the specification andclaims, range limitations may be combined and/or interchanged, suchranges are identified and include all the sub-ranges contained thereinunless context or language indicates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

In the descriptions above and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

What is claimed is:
 1. A system for screening chemical compounds fortheir ability to modulate the biological activity of a cell or acomponent of a cell, comprising: an assay device comprising amultiplicity of wells wherein each well is separated from other wellsand where said device comprises over 50,000 wells, a plurality of beadswhere a single bead is suitable for disposal in a single well, whereineach bead comprises a plurality of substantially identical bead-boundcompounds, wherein said bead-bound compounds are covalently linked tothe bead by a cleavable linker such that said compounds are releasablefrom said bead in a measurable dose dependent manner as part of anassay, said beads further comprising a plurality of substantiallyidentical bead-bound DNA barcodes linked to the bead (i) by a cleavablelinker or (ii) by a non-cleavable linker, wherein if the DNA barcodesare linked to the bead by a cleavable linker, the cleavable linker isorthogonal to the cleavable linker used to link the bead-bound compoundsto the bead, and wherein the DNA barcode identifies the compound.
 2. Thesystem of claim 1 further comprising a transfer device capable ofdispensing a single bead into a single well.
 3. The system of claim 1,wherein the transfer device is operated robotically, or manually or acombination of robotic and manual processes.
 4. The system of claim 2,wherein the transfer device employs magnetic attraction, electrostaticattraction or engineering principles based on size and gravity todeposit a single bead into a single well.
 5. The system of claim 2,wherein the transfer device further comprises at least one pipettecapable of transmitting the single bead, the at least one pipetteincluding a flexible tip.
 6. The system of claim 5, wherein the flexibletip of the pipette includes polyimide.
 7. The system of claim 5, whereinthe flexible tip extends along no more than 20% of a total length of thepipette.
 8. The system of claim 5, wherein the flexible tip extendsalong no more than 10% of a total length of the pipette.
 9. A method foridentifying a transcriptome change in a cell induced by a compound,wherein said compound is included in an assay of a combinatoriallibrary, the method comprising: a) generating an assay array whereinsaid assay array comprises: i) a plurality of wells wherein each well isseparated from other wells and each well comprises at least one cell ofinterest wherein assay array comprises over 50,000 wells, ii) aplurality of beads where a single bead wherein each bead comprises aplurality of same bead-bound compound such that each bead comprises aunique compound from said combinatorial library and each compound insaid library is selected as a potential drug candidate, and a pluralityof functionalized oligonucleotides, wherein said functionalizedoligonucleotide comprises an oligonucleotide portion that encodes thestructure of the unique compound or the synthetic steps used to makesaid unique compound and a RNA capturing element, wherein a single beadis disposed in a single well, b) contacting the cell in each confinedvolume with the compound released into the confined volume from the beadand maintaining said contact for a period sufficient to generate atranscriptome change in the RNA expressed by the cell in response to thesaid contacting; c) capturing RNA from the cell in each well by lysingthe cell and contacting the RNA with the RNA capturing element on saidbead; d) identifying the captured RNA from at least a portion of theplurality of beads and assessing any transcriptome change in saidcaptured RNA; and e) identifying the structure of the compound thatgenerated said transcriptome change.
 10. The method of claim 9, whereinsaid beads are added to the wells of said assay array using a transferdevice capable of dispensing a single bead into a single well.
 11. Themethod of claim 9, wherein the transfer device is operated robotically,or manually or a combination of robotic and manual processes.
 12. Themethod of claim 10, wherein the transfer device employs magneticattraction, electrostatic attraction or engineering principles based onsize and gravity to deposit a single bead into a single well.
 13. Themethod of claim 10, wherein the transfer device further comprises atleast one pipette capable of transmitting the single bead, the at leastone pipette including a flexible tip.
 14. The method of claim 13,wherein the flexible tip of the pipette includes polyimide.
 15. Themethod of claim 13, wherein the flexible tip extends along no more than20% of a total length of the pipette.
 16. The method of claim 13,wherein the flexible tip extends along no more than 10% of a totallength of the pipette.
 17. A system for screening chemical compounds fortheir ability to modulate the biological activity of a cell or acomponent of a cell, comprising: an assay device comprising amultiplicity of wells wherein each well is separated from other wells, aplurality of beads where a single bead is suitable for disposal in asingle well, wherein each bead comprises a plurality of substantiallyidentical bead-bound compounds, wherein said bead-bound compounds arecovalently linked to the bead by a cleavable linker such that saidcompounds are releasable from said bead in a measurable dose dependentmanner as part of an assay, said beads further comprising a plurality ofsubstantially identical bead-bound DNA barcodes linked to the bead (i)by a cleavable linker or (ii) by a non-cleavable linker, wherein if theDNA barcodes are linked to the bead by a cleavable linker, the cleavablelinker is orthogonal to the cleavable linker used to link the bead-boundcompounds to the bead, and wherein the DNA barcode identifies thecompound; and wherein each bead comprises at least about 10,000substantially identical DNA barcodes.
 18. The system of claim 17,further comprising a transfer device capable of dispensing a single beadinto a single well.
 19. The system of claim 17, wherein the transferdevice is operated robotically, or manually or a combination of roboticand manual processes.
 20. The system of claim 18, wherein the transferdevice employs magnetic attraction, electrostatic attraction orengineering principles based on size and gravity to deposit a singlebead into a single well.
 21. The system of claim 18, wherein thetransfer device further comprises at least one pipette capable oftransmitting the single bead, the at least one pipette including aflexible tip.
 22. The system of claim 21, wherein the flexible tip ofthe pipette includes polyimide.
 23. The system of claim 21, wherein theflexible tip extends along no more than 20% of a total length of thepipette.
 24. The system of claim 21, wherein the flexible tip extendsalong no more than 10% of a total length of the pipette.
 25. A methodfor identifying a transcriptome change in a cell induced by a compound,wherein said compound is included in an assay of a combinatoriallibrary, the method comprising: a) generating an assay array whereinsaid assay array comprises: i) a plurality of wells wherein each well isseparated from other wells and each well comprises at least one cell ofinterest, ii) a plurality of beads where a single bead wherein each beadcomprises a plurality of same bead-bound compound such that each beadcomprises a unique compound from said combinatorial library and eachcompound in said library is selected as a potential drug candidate, anda plurality of functionalized oligonucleotides, wherein saidfunctionalized oligonucleotide comprises a DNA barcode that encodes thestructure of the unique compound or the synthetic steps used to makesaid unique compound and a RNA capturing element, wherein a single beadis disposed in a single well, and wherein the DNA barcode identifies thecompound, and wherein each bead comprises at least about 10,000substantially identical DNA barcodes; b) contacting the cell in eachconfined volume with the compound released into the confined volume fromthe bead and maintaining said contact for a period sufficient togenerate a transcriptome change in the RNA expressed by the cell inresponse to the said contacting; c) capturing RNA from the cell in eachwell by lysing the cell and contacting the RNA with the RNA capturingelement on said bead; d) identifying the captured RNA from at least aportion of the plurality of beads and assessing any transcriptome changein said captured RNA; and e) identifying the structure of the compoundthat generated said transcriptome change.
 26. The method of claim 25,wherein said beads are added to the wells of said assay array using atransfer device capable of dispensing a single bead into a single well.27. The method of claim 25, wherein the transfer device is operatedrobotically, or manually or a combination of robotic and manualprocesses.
 28. The method of claim 26, wherein the transfer deviceemploys magnetic attraction, electrostatic attraction or engineeringprinciples based on size and gravity to deposit a single bead into asingle well.
 29. The method of claim 26, wherein the transfer devicefurther comprises at least one pipette capable of transmitting thesingle bead, the at least one pipette including a flexible tip.
 30. Themethod of claim 29, wherein the flexible tip of the pipette includespolyimide.
 31. The method of claim 28, wherein the flexible tip extendsalong no more than 20% of a total length of the pipette.
 32. The methodof claim 28, wherein the flexible tip extends along no more than 10% ofa total length of the pipette.